Target analyte detection using asymmetrical self-assembled monolayers

ABSTRACT

The present invention relates to the use asymmetric monolayer forming species and electroconduit forming species to detect target analytes.

This application is a continuation of U.S. Ser. No.: 09/847,113, filedMay 1, 2001, which claims the benefit of U.S. Ser. No. 60/201,026, filedMay 1, 2000 and is a continuation-in-part application of U.S. Ser. No.09/626,096, filed Jul. 26, 2000.

FIELD OF THE INVENTION

The present invention relates to the use asymmetric monolayer formingspecies and electroconduit forming species to detect target analytes.

BACKGROUND OF THE INVENTION

There are a number of assays and sensors for the detection of thepresence and/or concentration of specific substances in fluids andgases. Many of these rely on specific ligand/antiligand reactions as themechanism of detection. That is, pairs of substances (i.e. the bindingpairs or ligand/antiligands) are known to bind to each other, whilebinding little or not at all to other substances. This has been thefocus of a number of techniques that utilize these binding pairs for thedetection of the complexes. These generally are done by labeling onecomponent of the complex in some way, so as to make the entire complexdetectable, using, for example, radioisotopes, fluorescent and otheroptically active molecules, enzymes, etc.

Other assays rely on electronic signals for detection. Of particularinterest are biosensors. At least two types of biosensors are known;enzyme-based or metabolic biosensors and binding or bioaffinity sensors.See for example U.S. Pat. Nos. 4,713,347; 5,192,507; 4,920,047;3,873,267; and references disclosed therein. While some of these knownsensors use alternating current (AC) techniques, these techniques aregenerally limited to the detection of differences in bulk (ordielectric) impedance.

The use of self-assembled monolayers (SAMs) on surfaces for binding anddetection of biological molecules has recently been explored. See forexample WO98/20162; PCT US98/12430; PCT US98/12082; PCT US99/01705;PCT/US99/21683; PCT/US99/10104; PCT/US99/01703; PCT/US00/31233; U.S.Pat. Nos. 5,620,850; 6,197,515; 6,013,459; 6,013,170; and 6,065,573; andreferences cited therein.

Accordingly, it is an object of the invention to provide novel methodsand compositions for the electronic detection of target analytes usingself-assembled monolayers.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides compositions comprising metallic surfaces comprising asymmetricmonolayer forming species comprising two components. One of thecomponents is a standard monolayer forming species, such an alkyl chain.The other component is an electroconduit forming species. Electroconduitforming species are short chain alkyl groups, which may be branched.

In a further embodiment, the invention provides methods of detecting atarget analyte in a test sample comprising attaching said target analyteto a metallic surface comprising asymmetric monolayer forming speciesvia binding to a capture binding ligand. Recruitment linkers, or labelprobes are directly or indirectly attached to the target analyte to forman assay complex. The method further comprises detecting electrontransfer between an electron transfer moiety and an electrode.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1R depict a number of different compositions of the invention.FIG. 1A depicts I, also referred to as P290. FIG. 1B depicts II, alsoreferred to as P291. FIG. 1C depicts III, also referred to as W31. FIG.1D depicts IV, also referred to as N6. FIG. 1E depicts V, also referredto as P292. FIG. 1F depicts II, also referred to as C23. FIG. 1G depictsVII, also referred to as C15. FIG. 1H depicts VIII, also referred to asC95. FIG. 1I depicts Y63. FIG. 1J depicts another compound of theinvention. FIG. 1K depicts N11. FIG. 1L depicts C131, with aphosphoramidite group and a DMT protecting group. FIG. 1M depicts W38,also with a phosphoramidite group and a DMT protecting group. FIG. 1Ndepicts the commercially available moiety that enables “branching” tooccur, as its incorporation into a growing oligonucleotide chain resultsin addition at both the DMT protected oxygens. FIG. 10 depicts gIen,also with a phosphoramidite group and a DMT protecting group, thatserves as a non-nucleic acid linker. FIGS. 1A to 1G and 1J are shownwithout the phosphoramidite and protecting groups (i.e. DMT) that arereadily added.

FIGS. 2A, 2B and 2C depict some useful disulfide embodiments. FIG. 2Adepicts one example of a general class of an asymmetric monolayerforming species. FIG. 2B depicts two embodiments that were used togenerate the data shown in FIG. 2C. In FIG. 2C, M44 is a standardmonolayer forming species; the structure of M44 is shown in Figure

FIGS. 3A, 3B, 3C, 3D, 3E, 3F and 3G depict the synthesis of somedisulfide embodiments. FIG. 3A depicts the general synthesis; with R, R′and R″ being C1 to C20 alkyl or aromatic derivatives and B being anybase such as HaOH, KOH, LiOH or MOR, with M being a metal. FIG. 3B showsthe synthesis of H-phosphonate, FIGS. 3C and 3D show the synthesis ofthe CPG derivative, and FIG. 3E shows the synthesis of the insulatorCT105. 3F and 3G depict some cyclic disulfide embodiments.

FIGS. 4A, 4B and 4C depict three preferred embodiments for attaching atarget nucleic acid sequence to the electrode. FIG. 4A depicts a targetsequence 120 hybridized to a capture probe 100 linked via a attachmentlinker 106, which as outlined herein may be either a conductive oligomeror an insulator. The electrode 105 comprises a monolayer 107 comprisingasymmetric monolayer forming species, i.e., alkyl chains 400 andelectroconduit forming species 410 (which may be short branched alkylchains, short alkyl chains, or a mixture of branched and short alkylchains). As for all the embodiments depicted in the figures, n is aninteger of at least 1, although as will be appreciated by those in theart, the system may not utilize a capture probe at all (i.e. n is zero),although this is generally not preferred. The upper limit of n willdepend on the length of the target sequence and the requiredsensitivity. FIG. 4B depicts the use of a single capture extender probe110 with a first portion 111 that will hybridize to a first portion ofthe target sequence 120 and a second portion that will hybridize to thecapture probe 100. FIG. 4C depicts the use of two capture extenderprobes 110 and 130. The first capture extender probe 110 has a firstportion 111 that will hybridize to a first portion of the targetsequence 120 and a second portion 112 that will hybridize to a firstportion 102 of the capture probe 100. The second capture extender probe130 has a first portion 132 that will hybridize to a second portion ofthe target sequence 120 and a second portion 131 that will hybridize toa second portion 101 of the capture probe 100. As will be appreciated bythose in the art, while these systems depict nucleic acid targets, theseattachment configurations may be used with non-nucleic acid capturebinding ligands.

FIGS. 5A, 5B, 5C and 5D depict several embodiments of the invention.FIG. 5A is directed to the use of a capture binding ligand 200 attachedvia an attachment linker 106 to the electrode 105. Target analyte 210binds to the capture binding ligand 200, and a solution binding ligand22 with a directly attached recruitment linker 230 with ETMs 135. FIG.5B depicts a similar embodiment using an indirectly attached recruitmentlinker 145 that binds to a second portion 240 of the solution bindingligand 220. FIG. 5C depicts the use of an anchor ligand 100 (referred toherein as an anchor probe when the ligand comprises nucleic acid) tobind the capture binding ligand 200 comprising a portion 120 that willbind to the anchor probe 100. As will be appreciated by those in theart, any of the FIG. 4 embodiments may be used here as well. FIG. 5Ddepicts the use of an amplifier probe 145.

FIGS. 6A and 6B show two competitive type assays of the invention. FIG.6A utilizes the replacement of a target analyte 210 with a targetanalyte analog 310 comprising a directly attached recruitment linker145. As will be appreciated by those in the art, an indirectly attachedrecruitment linker can also be used, as shown in FIG. 6B. FIG. 6B showsa competitive assay wherein the target analyte 210 and the targetanalyte analog 310 attached to the surface compete for binding of asolution binding ligand 220 with a directly attached recruitment linker145 (again, an indirectly attached recruitment linker can also be used,as shown in FIG. 5B). In this case, a loss of signal may be seen.

FIGS. 7A-7R depict nucleic acid detection systems. FIGS. 7A and 7B havethe target sequence 5 containing the ETMs 6; as discussed herein, thesemay be added enzymatically, for example during a PCR reaction usingnucleotides modified with ETMs, resulting in essentially randomincorporation throughout the target sequence, or added to the terminusof the target sequence. FIG. 7A shows attachment of a capture probe 10to the electrode 20 via a linker 15, which as discussed herein can beeither a conductive oligomer or an insulator. The target sequence 5contains ETMs 6. FIG. 7B depicts the use of a capture extender probe 11,comprising a first portion 12 that hybridizes to a portion of the targetsequence and a second portion 13 that hybridizes to the capture probe10.

FIG. 7C depicts the use of two different capture probes 10 and 10′, thathybridize to different portions of the target sequence 5. As will beappreciated by those in the art, the 5′-3′ orientation of the twocapture probes in this embodiment is different.

FIGS. 7D to 7H depict the use of label probes 40 that hybridize directlyto the target sequence 5. FIG. 7D shows the use of a label probe 40,comprising a first portion 41 that hybridizes to a portion of the targetsequence 5, a second portion 42 that hybridizes to the capture probe 10and a recruitment linker 50 comprising ETMs 6. A similar embodiment isshown in FIG. 7E, where the label probe 40 has an additional recruitmentlinker 50. FIG. 7F depicts a label probe 40 comprising a first portion41 that hybridizes to a portion of the target sequence 5 and arecruitment linker 50 with attached ETMs 6. The parentheses highlightthat for any particular target sequence 5 more than one label probe 40may be used, with n being an integer of at least 1. FIG. 7G depicts theuse of the FIG. 7E label probe structures but includes the use of asingle capture extender probe 11, with a first portion 12 thathybridizes to a portion of the target sequence and a second portion 13that hybridizes to the capture probe 10. FIG. 7H depicts the use of theFIG. 7F label probe structures but utilizes two capture extender probes11 and 16. The first capture extender probe 11 has a first portion 12that hybridizes to a portion of the target sequence 5 and a secondportion 13 that hybridizes to a first portion 14 of the capture probe10. The second capture extender probe 16 has a first portion 18 thathybridizes to a second portion of the target sequence 5 and a secondportion 17 that hybridizes to a second portion 19 of the capture probe10.

FIGS. 7I, 7J and 7K depict systems utilizing label probes 40 that do nothybridize directly to the target, but rather to amplifier probes. Thusthe amplifier probe 60 has a first portion 65 that hybridizes to thetarget sequence 5 and at least one second portion 70, i.e. the amplifiersequence, that hybridizes to the first portion 41 of the label probe.

FIGS. 7L, 7M and 7N depict systems that utilize a first label extenderprobe 80. In these embodiments, the label extender probe 80 has a firstportion 81 that hybridizes to a portion of the target sequence 5, and asecond portion 82 that hybridizes to the first portion 65 of theamplifier probe 60.

FIG. 7O depicts the use of two label extender probes 80 and 90. Thefirst label extender probe 80 has a first portion 81 that hybridizes toa portion of the target sequence 5, and a second portion 82 thathybridizes to a first portion 62 of the amplifier probe 60. The secondlabel extender probe 90 has a first portion 91 that hybridizes to asecond portion of the target sequence 5 and a second portion 92 thathybridizes to a second portion 61 of the amplifier probe 60.

FIG. 7P depicts a system utilizing a label probe 40 hybridizing to theterminus of a target sequence 5.

FIGS. 7Q and 7R depict systems that utilizes multiple label probes. Thefirst portion 41 of the label probe 40 can hybridize to all (FIG. 7R) orpart (FIG. 7Q) of the recruitment linker 50. FIG. 8 depicts a detectionsystem with a label probe labeled with multiple ETMs, in which a firstportion hybridizes to a portion of a target sequence and a capture probethat hybridizes to a different portion of the target sequence.

FIG. 9 depicts the chemical structures of a standard monolayer formingspecies, M44 and two asymmetrical monolayer forming species, CT99 andCT105.

FIG. 10 depicts an example of a layout for an array chip with sensorpads. FIG. 11 depicts the electrochemical response of asymmetricmonolayer forming species vs a standard monolayer forming species in adirect assay using a 2 N6 ferrocene signaling probe.

FIG. 12 depicts the electrochemical response of asymmetric monolayerforming species vs a standard monolayer forming species a sandwich assayusing 8 N6 ferrocene.

FIG. 13 depicts the electrochemical response of asymmetric monolayerforming species versus a standard monolayer forming species a sandwichassay using a 20 C23 type ferrocene.

FIG. 14 depicts the electrochemical response of asymmetric monolayerforming species vs a standard monolayer forming species a sandwich assayusing a 54 C23 type ferrocene.

FIG. 15 depicts the nonspecific binding of asymmetric monolayer formingspecies versus a standard monolayer forming species in a direct assay at1000 Hz and 4^(th) harmonics.

FIG. 16 depicts the nonspecific binding of asymmetric monolayer formingspecies versus a standard monolayer forming species in a sandwich assayat 1000 Hz and 4^(th) harmonics.

FIG. 17 depicts a monolayer comprising insulators only (i.e. M44) and amonolayer comprising asymmetric monolayer forming species (i.e. CT105).

FIG. 18 depicts the frequency response for D1085 of two N6 ferrocenes.

FIG. 19 depicts the frequency response for a sandwich assay of an 8ferrocene system.

FIG. 20 depicts the frequency response for a sandwich assay of an 20ferrocene system.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the electronic detection ofanalytes. Previous work, described in PCT US97/20014, is directed to thedetection of nucleic acids, and utilizes nucleic acids covalentlyattached to electrodes using conductive oligomers, i.e. chemical“wires”. Upon formation of double stranded nucleic acids containingelectron transfer moieties (ETMs), electron transfer can proceed throughthe stacked ri-orbitals of the heterocyclic bases to the electrode, thusenabling electronic detection of target nucleic acids (termed “mechanism1”). In the absence of the stacked n-orbitals, i.e. when the targetstrand is not present, electron transfer is negligible, thus allowingthe use of the system as an assay. This previous work also reported onthe use of self-assembled monolayers (SAMs) to electronically shield theelectrodes from solution components and significantly decrease theamount of non-specific binding to the electrodes.

Alternatively, the ETM can be detected, not necessarily via electrontransfer through nucleic acid, but rather can be directly detected on anelectrode comprising a SAM; that is, the electrons from the ETMs neednot travel through the stacked n orbitals in order to generate a signal.As above, in this embodiment, the detection electrode preferablycomprises a self-assembled monolayer (SAM) that serves to shield theelectrode from redox-active species in the sample. In this embodiment,the presence of ETMs on the surface of a SAM, that has been formulatedto comprise slight “defects” (sometimes referred to herein as“microconduits”, “nanoconduits” or “electroconduits”) can be directlydetected. This basic idea is termed “mechanism-2” herein. Essentially,the electroconduits allow particular ETMs access to the surface. Uponbinding of a target analyte to a binding species on the surface, arecruitment linker or label probe comprising at least one ETM is broughtto the surface, and detection of the ETM can proceed. Thus, the role ofthe target analyte and-binding species is to provide specificity for arecruitment of ETMs to the surface, where they can be detected using theelectrode. The role of the asymmetric monolayer species comprising thedefects is to allow contact of the ETM with the electronic surface ofthe electrode, while still providing the benefits of shielding theelectrode from solution components and reducing the amount ofnon-specific binding to the electrodes. See, for example, WO98/20162;PCT US98/12430; PCT US98/12082; PCT US99/01705; PCT/US99/21683;PCT/US99/10104; PCT/US99/01703; PCT/US00/20476; PCT/US00/31233; U.S.Pat. Nos. 5,620,850; 6,197,515; 6,013,459; 6,013,170; and 6,065,573; andU.S. Ser. Nos. 09/660,374; and, 09/135,183 and references cited therein.

Thus, the present invention is directed to novel compositions ofmonolayer forming species that are thought to form electroconduits; thatis, the present invention is directed to the use of monolayerscomprising asymmetric monolayer forming species (“AMFS”). As describedmore fully below, AMFS comprise two components, usually linked by adisulfide bridge, at least one of which is a standard monolayer formingspecies such as an alkyl chain, and the other is a shorter species, forexample a shorter alkyl chain or a short branched chain. These twoelements are put down together, for example by attaching them as adisulfide moiety that then is used to form a monolayer on a metallicsurface such as gold. The “shorter” element thus, is thought to form anelectroconduit, and protect the surface from redox-active species insolution.

Without being bound by theory, it should be noted that the configurationof the electroconduit depends in part on the ETM chosen. For example,the use of relatively hydrophobic ETMs allows the use of hydrophobicelectroconduit forming species, which effectively exclude hydrophilic orcharged ETMs. Similarly, the use of more hydrophilic or charged speciesin the SAM may serve to exclude hydrophobic ETMs.

Asymmetric monolayer forming species preferably comprise a mixture ofinsulators and electroconduit forming species, although conductiveoligomers as either component, also may be included. Preferably, theinsulators are long chain alkyl groups from about 7 to 20 carbons inlength which are covalently attached to a metallic surface via a linkermoiety such as sulfur. Electroconduit forming species include alkylgroups, phenyl-acetylene-polyethylene glycol species, and branched alkylgroups. In addition asymmetric monolayer forming species includeasymmetrical SAM-forming disulfide species such as depicted in FIG. 3.

The invention can be generally described as follows, with a number ofpossible embodiments depicted in the Figures. In a preferred embodiment,as depicted in FIG. 5, an electrode comprising an asymmetric monolayerforming species comprising insulators (preferably a long chain alkylgroup), an electroconduit forming species, and a covalently attachedtarget analyte binding ligand (frequently referred to herein as a“capture binding ligand”) is made. The target analyte is added, whichbinds to the support-bound binding ligand. A solution binding ligand isadded, which may be the same or different from the first binding ligand,which can also bind to the target analyte, forming a “sandwich” ofsorts. The solution binding ligand either comprises a recruitment linkercontaining ETMs, or comprises a portion that will either directly orindirectly bind a recruitment linker containing the ETMs. This“recruitment” of ETMs to the surface of the monolayer allows electronicdetection via electron transfer between the ETM and the electrode. Inthe absence of the target analyte, the recruitment linker is eitherwashed away or not in sufficient proximity to the surface to allowdetection.

For example, when both the target analyte and the capture binding ligand(generally referred to herein as a “capture probe” when it is a nucleicacid) are nucleic acids, a preferred embodiment is shown in FIG. 8. Inthis embodiment, the surface comprises an AMFS and a capture probe. Afirst portion of the target sequence hybridizes to the capture probe,and a label probe, comprising a recruitment linker comprising ETMs,hybridizes to a second portion of the target sequence.

In an alternate preferred embodiment, as depicted in FIG. 6, acompetitive binding type assay is run. In this embodiment, the targetanalyte in the sample is replaced by a target analyte analog as isdescribed below and generally known in the art. The analog comprises adirectly or indirectly attached recruitment linker comprising at leastone ETM. The binding of the analog to the capture binding ligandrecruits the ETM to the surface and allows detection based on electrontransfer between the ETM and the electrode.

In an additional preferred embodiment, as depicted in FIG. 6B, acompetitive assay wherein the target analyte and a target analyte analogattached to the surface compete for binding of a solution binding ligandwith a directly or indirectly attached recruitment linker. In this case,a loss of signal may be seen.

Accordingly, the present invention provides methods and compositionsuseful in the detection of target analytes. As will be appreciated bythose in the art, the sample solution may comprise any number of things,including, but not limited to, bodily fluids (including, but not limitedto, blood, urine, serum, lymph, saliva, anal and vaginal secretions,perspiration and semen, of virtually any organism, with mammaliansamples being preferred and human samples being particularly preferred);environmental samples (including, but not limited to, air, agricultural,water and soil samples); biological warfare agent samples; researchsamples (i.e. in the case of nucleic acids, the sample may be theproducts of an amplification reaction, including both target and signalamplification as is generally described in PCT/US99/01705, such as PCRamplification reaction); purified samples, such as purified genomic DNA,RNA, proteins, etc.; raw samples (bacteria, virus, genomic DNA, etc.).As will be appreciated by those in the art, virtually any experimentalmanipulation may have been done on the sample.

By “target analyte” or “analyte” or grammatical equivalents herein ismeant any molecule or compound to be detected and that can bind to abinding species, defined below. Suitable analytes include, but notlimited to, small chemical molecules such as environmental or clinicalchemical or pollutant or biomolecule, including, but not limited to,pesticides, insecticides, toxins, therapeutic and abused drugs,hormones, antibiotics, antibodies, organic materials, etc. Suitablebiomolecules include, but are not limited to, proteins (includingenzymes, immunoglobulins and glycoproteins), nucleic acids, lipids,lectins, carbohydrates, hormones, whole cells (including procaryotic(such as pathogenic bacteria) and eucaryotic cells, including mammaliantumor cells), viruses, spores, etc. Particularly preferred analytes areproteins including enzymes; drugs, cells; antibodies; antigens; cellularmembrane antigens and receptors (neural, hormonal, nutrient, and cellsurface receptors) or their ligands.

By “proteins” or grammatical equivalents herein is meant proteins,oligopeptides and peptides, and analogs, including proteins containingnon-naturally occurring amino acids and amino acid analogs, andpeptidomimetic structures.

As will be appreciated by those in the art, a large number of analytesmay be detected using the present methods; basically, any target analytefor which a binding ligand, described below, may be made may be detectedusing the methods of the invention.

By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 1 10:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); those with bicyclicstructures including locked nucleic acids, Koshkin et al., J. Am. Chem.Soc. 120:13252-3 (1998); non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew.Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem.Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597(1994); Chapters 2 and 3, ASC Symposium Series 580, “CarbohydrateModifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook;Mesmaeker et al., Bioorganic & Medicinal Chem. Left. 4:395 (1994); Jeffset al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Left. 37:743(1996)) and non-ribose backbones, including those described in U.S. Pat.Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S.Sanghui and P. Dan Cook. Nucleic acids containing one or morecarbocyclic sugars are also included within the definition of nucleicacids (see Jenkins et al., Chem. Soc. Rev. (1995) pp169-176). Severalnucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997page 35. All of these references are hereby expressly incorporated byreference. These modifications of the ribose-phosphate backbone may bedone to facilitate the addition of ETMs, or to increase the stabilityand half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids. Thisresults in two advantages. First, the PNA backbone exhibits improvedhybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch.With the non-ionic PNA backbone, the drop is closer to 7-9° C.Similarly, due to their non-ionic nature, hybridization of the basesattached to these backbones is relatively insensitive to saltconcentration. This is particularly advantageous in the systems of thepresent invention, as a reduced salt hybridization solution has a lowerFaradaic current than a physiological salt solution (in the range of 150mM).

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. A preferred embodimentutilizes isocytosine and isoguanine in nucleic acids designed to becomplementary to other probes, rather than target sequences, as thisreduces non-specific hybridization, as is generally described in U.S.Pat. No. 5,681,702. As used herein, the term “nucleoside” includesnucleotides as well as nucleoside and nucleotide analogs, and modifiednucleosides such as amino modified nucleosides. In addition,“nucleoside” includes non-naturally occuring analog structures. Thus forexample the individual units of a peptide nucleic acid, each containinga base, are referred to herein as a nucleoside.

As will be appreciated by those in the art, a large number of analytesmay be detected using the present methods; basically, any target analytefor which a binding ligand, described below, may be made may be detectedusing the methods of the invention.

Accordingly, the present invention provides methods and compositionsuseful in the detection of target analytes. In a preferred embodiment,the compositions comprise a substrate comprising a metallic surfacecomprising an asymmetric monolayer forming species.

By “substrate” or “solid support” or other grammatical equivalentsherein is meant any material that can be modified to contain discreteindividual sites appropriate for the attachment or association of targetanalytes. The substrate can comprise a wide variety of materials, aswill be appreciated by those in the art, with printed circuit board(PCB) materials being particularly preferred. Other suitable substratesinclude, but are not limited to, metal surfaces such as gold, electrodesas defined below, glass and modified or functionalized glass, plastics(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes),Teflon™, fiberglass, GETEK (a blend of polypropylene oxide andfiberglass), etc.), polysaccharides, nylon or nitrocellulose, resins,ceramics, mica, silica or silica-based materials including silicon andmodified silicon, carbon, metals, inorganic glasses, and a variety ofother polymers.

In general, preferred materials include printed circuit board materials.Circuit board materials are those that comprise an insulating substratethat is coated with a conducting layer and processed using lithographytechniques, particularly photolithography techniques, to form thepatterns of electrodes and interconnects (sometimes referred to in theart as interconnections or leads). The insulating substrate isgenerally, but not always, a polymer. As is known in the art, one or aplurality of layers may be used, to make either “two dimensional” (e.g.all electrodes and interconnections in a plane) or “three dimensional”(wherein the electrodes are on one surface and the interconnects may gothrough the board to the other side) boards. Three dimensional systemsfrequently rely on the use of drilling or etching, followed byelectroplating with a metal such as copper, such that the “throughboard” interconnections are made. Circuit board materials are oftenprovided with a foil already attached to the substrate, such as a copperfoil, with additional copper added as needed (for example forinterconnections), for example by electroplating. The copper surface maythen need to be roughened, for example through etching, to allowattachment of the adhesion layer.

The substrates comprise one or more metallic surfaces, preferablyelectrodes. By “metallic surface” or other grammatical equivalentsherein is meant any material that can be modified to contain discreteindividual sites appropriate for the attachment or association of targetanalytes. Preferred metallic surfaces include, but are not limited to,gold, indium tin oxide, and electrodes.

In a preferred embodiment, the metallic surface is an electrode. By“electrode” herein is meant a composition, which, when connected to anelectronic device, is able to sense a current or charge and convert itto a signal. Thus, an electrode is an ETM as described herein. Preferredelectrodes are known in the art and include, but are not limited to,certain metals and their oxides, including gold; platinum; palladium;silicon; aluminum; metal oxide electrodes including platinum oxide,titanium oxide, tin oxide, indium tin oxide, palladium oxide, siliconoxide, aluminum oxide, molybdenum oxide (Mo₂O₆), tungsten oxide (WO₃)and ruthenium oxides; and carbon (including glassy carbon electrodes,graphite and carbon paste). Preferred electrodes include gold, silicon,carbon and metal oxide electrodes, with gold being particularlypreferred.

The substrate may also include arrays, i.e. wherein there is a matrix ofaddressable detection electrodes (herein generally referred to “pads”,“addresses” or “micro-locations”). By “array” herein is meant aplurality of capture ligands in an array format; the size of the arraywill depend on the composition and end use of the array. Arrayscontaining from about 2 different capture ligands to many thousands canbe made. Generally, the array will comprise from two to as many as100,000 or more, depending on the size of the electrodes, as well as theend use of the array. Preferred ranges are from about 2 to about 10,000,with from about 5 to about 1000 being preferred, and from about 10 toabout 100 being particularly preferred. In some embodiments, thecompositions of the invention may not be in array format; that is, forsome embodiments, compositions comprising a single capture ligand may bemade as well. In addition, in some arrays, multiple substrates may beused, either of different or identical compositions. Thus for example,large arrays may comprise a plurality of smaller substrates.

The electrodes described herein are depicted as a flat surface, which isonly one of the possible conformations of the electrode and is forschematic purposes only. The conformation of the electrode will varywith the detection method used. For example, flat planar electrodes maybe preferred for optical detection methods, or when arrays of nucleicacids are made, thus requiring addressable locations for both synthesisand detection. Alternatively, for single probe analysis, the electrodemay be in the form of a tube, with the SAMs comprising AMFS and nucleicacids bound to the inner surface. This allows a maximum of surface areacontaining the nucleic acids to be exposed to a small volume of sample.

The electrode comprises asymmetric monolayer forming species. By“monolayer” or “self-assembled monolayer” or “SAM” herein is meant arelatively ordered assembly of molecules spontaneously chemisorbed on asurface, in which the molecules are oriented approximately parallel toeach other and roughly perpendicular to the surface. Each of themolecules includes a functional group that adheres to the surface, and aportion that interacts with neighboring molecules in the monolayer toform the relatively ordered array. A “mixed” monolayer comprises aheterogeneous monolayer, that is, where at least two different moleculesmake up the monolayer. As outlined herein, the use of a monolayerreduces the amount of non-specific binding of biomolecules to thesurface, and, in the case of nucleic acids, increases the efficiency ofoligonucleotide hybridization as a result of the distance of theoligonucleotide from the electrode. Thus, a monolayer facilitates themaintenance of the target analyte away from the electrode surface. Inaddition, a monolayer serves to keep charge carriers away from thesurface of the electrode. Thus, this layer helps to prevent electricalcontact between the electrodes and the ETMs, or between the electrodeand charged species within the solvent. Such contact can result in adirect “short circuit” or an indirect short circuit via charged specieswhich may be present in the sample. Accordingly, the monolayer ispreferably tightly packed in a uniform layer on the electrode surface,such that a minimum of “holes” exist. The monolayer thus serves as aphysical barrier to block solvent accessibility to the electrode.

In a preferred embodiment, the AMFS comprises a standard monolayerforming species comprises a standard monolayer forming species. Bystandard monolayer forming species herein is meant an alkyl chain,preferably linear, from about 7 to 20 carbons in length.

In a preferred embodiment, the AFMS comprise insulator moieties. By“insulator” herein is meant a substantially nonconducting oligomer,preferably linear. By “substantially nonconducting” herein is meant thatthe insulator will not transfer electrons at or above 100 Hz when an ACvoltage is applied. The rate of electron transfer through the insulatoris preferably slower than the rate through the conductive oligomersdescribed herein.

In a preferred embodiment, the insulators have a conductivity, S, ofabout 10⁻⁷Ω⁻¹cm⁻¹ or lower, with less than about 10⁻⁸Ω⁻¹cm⁻¹ beingpreferred. See generally Gardner et al., supra.

Generally, insulators are alkyl or heteroalkyl oligomers or moietieswith sigma bonds, although any particular insulator molecule may containaromatic groups or one or more conjugated bonds. By “heteroalkyl” hereinis meant an alkyl group that has at least one heteroatom, i.e. nitrogen,oxygen, sulfur, phosphorus, silicon or boron included in the chain.Alternatively, the insulator may be quite similar to a conductiveoligomer with the addition of one or more heteroatoms or bonds thatserve to inhibit or slow, preferably substantially, electron transfer.

Suitable insulators are known in the art, and include, but are notlimited to, —(CH₂)_(n)—, —(CRH)_(n)—, and —(CR₂)_(n)—, ethylene glycolor derivatives using other heteroatoms in place of oxygen, i.e. nitrogenor sulfur (sulfur derivatives are not preferred when the electrode isgold).

The insulators may be substituted with R groups as defined below toalter the packing of the moieties or conductive oligomers on anelectrode, the hydrophilicity or hydrophobicity of the insulator, andthe flexibility, i.e. the rotational, torsional or longitudinalflexibility of the insulator. For example, branched alkyl groups may beused. Similarly, the insulators may contain terminal groups, as outlinedabove, particularly to influence the surface of the monolayer.

The length of the insulator may vary. Preferably the insulator is astraight chain alkyl group, comprising (CH₂)_(n) and (OCH₂CH₂)_(n)groups and a terminal OH group. The integer, n, will vary, but generallywill be from about 7 to 20 for (CH₂)_(n) and from about 0 to 10 for(OCH₂CH₂)_(n).

In a preferred embodiment, the asymmetric monolayer forming speciescomprises a disulfide group which links a monolayer forming species,such as an insulator, and an asymmetric group. Preferably, theasymmetric group is an electroconduit forming species (EFS). By“electroconduit-forming species” or “EFS” herein is meant a moleculethat is capable of generating sufficient electroconduits in a monolayer,generally of insulators such as alkyl groups, to allow detection of ETMsat the surface. In general, EFS have one or more of the followingqualities: they may be relatively rigid molecules, for example ascompared to an alkyl chain; they may attach to the electrode surfacewith a geometry different from the other monolayer forming species (forexample, alkyl chains attached to gold surfaces with thiol groups arethought to attach at roughly 45° angles, and phenyl-acetylene chainsattached to gold via thiols are thought to go down at 90° angles); theymay have a structure that sterically interferes or interrupts theformation of a tightly packed monolayer, for example through theinclusion of branching groups such as alkyl groups, or the inclusion ofhighly flexible species, such as polyethylene glycol units; or they maybe capable of being activated to form electroconduits; for example,photoactivatible species that can be selectively removed from thesurface upon photoactivation, leaving electroconduits.

Preferred EFS include conductive oligomers, as defined below, andphenyl-acetylene-polyethylene glycol species, and branched alkyl groups.

In a preferred embodiment, the EFS is an alkyl group as defined below.If the EFS is a straight chain alkyl group, 1 to 6 carbon atoms arepreferred.

In a preferred embodiment, the EFS is a branched chain alkyl group,substituted with one or more substitution moieties “R” as defined below.It may be branched at one or more positions. The EFS may be directlyattached to an attachment linker as defined below. Alternatively, theEFS may be attached to the attachment linker via a (CH₂)_(n) group,wherein n is an integer from 1 to 4.

In one embodiment, the AFMS has the structure depicted in Structure 44:

Structure 44

-   -   EFS—S—S—I

In Structure 44, I represents an insulator moiety as defined within, EFSis an electroconduit moiety as defined within and S represents a S atom.

In a preferred embodiment, the AFMS has the structure depicted inStructure 45:

In this embodiment, n is an integer from 7-16, m is an integer from 0-7and o is an integer from 0 to 4. In Structure 45, R1, R2 and R3 may eachbe independently selected from the group consisting of hydrogen, alkylgroups including cycloalkyl, alchol groups, amine groups, amido, ester,phosphorus moieties, and aryl groups including substituted aryl andheteroaryl.

In a preferred embodiment, the AFMS has the structure depicted inStructure 46:

In a preferred embodiment, the AFMS has the structure depicted inStructure 47:

In one embodiment, in addition to the AFMS, the monolayer comprisesinsulators.

In one embodiment, in addition to the AFMS, the monolayer comprisesconductive oligomers. By “conductive oligomer” herein is meant asubstantially conducting oligomer, preferably linear, some embodimentsof which are referred to in the literature as “molecular wires”. By“substantially conducting” herein is meant that the oligomer is capableof transfering electrons at 100 Hz. Generally, the conductive oligomerhas substantially overlapping n-orbitals, i.e. conjugated n-orbitals, asbetween the monomeric units of the conductive oligomer, although theconductive oligomer may also contain one or more sigma (a) bonds.Additionally, a conductive oligomer may be defined functionally by itsability to inject or receive electrons into or from an associated ETM.Furthermore, the conductive oligomer is more conductive than theinsulators as defined herein. Additionally, the conductive oligomers ofthe invention are to be distinguished from electroactive polymers, thatthemselves may donate or accept electrons.

In a preferred embodiment, the conductive oligomers have a conductivity,S, of from between about 10⁻⁶ to about 10⁴Ω⁻¹cm⁻¹, with from about 10⁻⁵to about 10³Ω⁻¹cm⁻¹ being preferred, with these S values beingcalculated for molecules ranging from about 20 A to about 200 A. Asdescribed below, insulators have a conductivity S of about 10⁻⁷Ω⁻¹cm⁻¹or lower, with less than about 10⁻⁸Ω⁻¹cm⁻¹ being preferred. Seegenerally Gardner et al., Sensors and Actuators A 51 (1995) 57-66,incorporated herein by reference.

Desired characteristics of a conductive oligomer include highconductivity, sufficient solubility in organic solvents and/or water forsynthesis and use of the compositions of the invention, and preferablychemical resistance to reactions that occur i) during nucleic acidsynthesis (such that nucleosides containing the conductive oligomers maybe added to a nucleic acid synthesizer during the synthesis of thecompositions of the invention), ii) during the attachment of theconductive oligomer to an electrode, or iii) during hybridizationassays. In addition, conductive oligomers that will promote theformation of self-assembled monolayers are preferred.

The oligomers of the invention comprise at least two monomeric subunits,as described herein. As is described more fully below, oligomers includehomo- and hetero-oligomers, and include polymers.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 1:

As will be understood by those in the art, all of the structuresdepicted herein may have additional atoms or structures; i.e. theconductive oligomer of Structure 1 may be attached to ETMs, such aselectrodes, transition metal complexes, organic ETMs, and metallocenes,and to nucleic acids, or to several of these. Unless otherwise noted,the conductive oligomers depicted herein will be attached at the leftside to an electrode; that is, as depicted in Structure 1, the left “Y”is connected to the electrode as described herein. If the conductiveoligomer is to be attached to a nucleic acid, the right “Y”, if present,is attached to the nucleic acid, either directly or through the use of alinker, as is described herein.

In this embodiment, Y is an aromatic group, n is an integer from 1 to50, g is either 1 or zero, e is an integer from zero to 10, and m iszero or 1. When g is 1, B-D is a bond able to conjugate with neighboringbonds (herein referred to as a “conjugated bond”), preferably selectedfrom acetylene, B-D D is a conjugated bond, preferably selected fromacetylene, alkene, substituted alkene, amide, azo, —C═N— (including—N═C—, —CR═N— and —N═CR—), —Si═Si—, and —Si═C— (including —C═Si—,—Si═CR— and —CR═Si—). When g is zero, e is preferably 1, D is preferablycarbonyl, or a heteroatom moiety, wherein the heteroatom is selectedfrom oxygen, sulfur, nitrogen, silicon or phosphorus. Thus, suitableheteroatom moieties include, but are not limited to, —NH and —NR,wherein R is as defined herein; substituted sulfur; sulfonyl (—SO₂—)sulfoxide (—SO—); phosphine oxide (—PO— and —RPO—); and thiophosphine(—PS— and —RPS—). However, when the conductive oligomer is to beattached to a gold electrode, as outlined below, sulfur derivatives arenot preferred.

By “aromatic group” or grammatical equivalents herein is meant anaromatic monocyclic or polycyclic hydrocarbon moiety generallycontaining 5 to 14 carbon atoms (although larger polycyclic ringsstructures may be made) and any carbocylic ketone or thioketonederivative thereof, wherein the carbon atom with the free valence is amember of an aromatic ring. Aromatic groups include arylene groups andaromatic groups with more than two atoms removed. For the purposes ofthis application aromatic includes heterocycle. “Heterocycle” or“heteroaryl” means an aromatic group wherein 1 to 5 of the indicatedcarbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen,sulfur, phosphorus, boron and silicon wherein the atom with the freevalence is a member of an aromatic ring, and any heterocyclic ketone andthioketone derivative thereof. Thus, heterocycle includes thienyl,furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl,isoquinolyl, thiazolyl, imidozyl, etc.

Importantly, the Y aromatic groups of the conductive oligomer may bedifferent, i.e. the conductive oligomer may be a heterooligomer. Thatis, a conductive oligomer may comprise a oligomer of a single type of Ygroups, or of multiple types of Y groups.

The aromatic group may be substituted with a substitution group,generally depicted herein as R. R groups may be added as necessary toaffect the packing of the conductive oligomers, i.e. R groups may beused to alter the association of the oligomers in the monolayer. Rgroups may also be added to 1) alter the solubility of the oligomer orof compositions containing the oligomers; 2) alter the conjugation orelectrochemical potential of the system; and 3) alter the charge orcharacteristics at the surface of the monolayer.

In a preferred embodiment, when the conductive oligomer is greater thanthree subunits, R groups are preferred to increase solubility whensolution synthesis is done. However, the R groups, and their positions,are chosen to minimally effect the packing of the conductive oligomerson a surface, particularly within a monolayer, as described below. Ingeneral, only small R groups are used within the monolayer, with largerR groups generally above the surface of the monolayer. Thus for examplethe attachment of methyl groups to the portion of the conductiveoligomer within the monolayer to increase solubility is preferred, withattachment of longer alkoxy groups, for example, C3 to C10, ispreferably done above the monolayer surface. In general, for the systemsdescribed herein, this generally means that attachment of stericallysignificant R groups is not done on any of the first two or threeoligomer subunits, depending on the average length of the moleculesmaking up the monolayer.

Suitable R groups include, but are not limited to, hydrogen, alkyl,alcohol, aromatic, amino, amido, nitro, ethers, esters, aldehydes,sulfonyl, silicon moieties, halogens, sulfur containing moieties,phosphorus containing moieties, and ethylene glycols. In the structuresdepicted herein, R is hydrogen when the position is unsubstituted. Itshould be noted that some positions may allow two substitution groups, Rand R′, in which case the R and R′ groups may be either the same ordifferent. By “alkyl group” or grammatical equivalents herein is meant astraight or branched chain alkyl group, with straight chain alkyl groupsbeing preferred. If branched, it may be branched at one or morepositions, and unless specified, at any position. The alkyl group mayrange from about 1 to about 30 carbon atoms (C1-C30), with a preferredembodiment utilizing from about 1 to about 20 carbon atoms (C1-C20),with about C1 through about C12 to about C15 being preferred, and C1 toC5 being particularly preferred, although in some embodiments the alkylgroup may be much larger. Also included within the definition of analkyl group are cycloalkyl groups such as C5 and C6 rings, andheterocyclic rings with nitrogen, oxygen, sulfur or phosphorus. Alkylalso includes heteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen,and silicone being preferred. Alkyl includes substituted alkyl groups.By “substituted alkyl group” herein is meant an alkyl group furthercomprising one or more substitution moieties “R”, as defined above.

By “amino groups” or grammatical equivalents herein is meant —NH₂, —NHRand —NR₂ groups, with R being as defined herein.

By “nitro group” herein is meant an —NO₂ group.

By “sulfur containing moieties” herein is meant compounds containingsulfur atoms, including but not limited to, thia-, thio- and sulfo-compounds, thiols (—SH and —SR), and sulfides (—RSR—). By “phosphoruscontaining moieties” herein is meant compounds containing phosphorus,including, but not limited to, phosphines and phosphates. By “siliconcontaining moieties” herein is meant compounds containing silicon.

By “ether” herein is meant an —O—R group. Preferred ethers includealkoxy groups, with —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ being preferred.

By “ester” herein is meant a —COOR group.

By “halogen” herein is meant bromine, iodine, chlorine, or fluorine.Preferred substituted alkyls are partially or fully halogenated alkylssuch as CF₃, etc.

By “aldehyde” herein is meant —RCHO groups.

By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH.

By “amido” herein is meant —RCONH— or RCONR— groups.

By “ethylene glycol” or “(poly)ethylene glycol” herein is meant a—(O—CH₂—CH₂)_(n)— group, although each carbon atom of the ethylene groupmay also be singly or doubly substituted, i.e. —(O—CR₂—CR₂)_(n)—, with Ras described above. Ethylene glycol derivatives with other heteroatomsin place of oxygen (i.e. —(N—CH₂—CH₂)_(n)— or —(S—CH₂—CH₂)_(n)—, or withsubstitution groups) are also preferred.

Preferred substitution groups include, but are not limited to, methyl,ethyl, propyl, alkoxy groups such as —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ andethylene glycol and derivatives thereof.

Preferred aromatic groups include, but are not limited to, phenyl,naphthyl, naphthalene, anthracene, phenanthroline, pyrole, pyridine,thiophene, porphyrins, and substituted derivatives of each of these,included fused ring derivatives.

In the conductive oligomers depicted herein, when g is 1, B-D is a bondlinking two atoms or chemical moieties. In a preferred embodiment, B-Dis a conjugated bond, containing overlapping or conjugated n-orbitals.

Preferred B-D bonds are selected from acetylene (—C≡C—, also calledalkyne or ethyne), alkene (—CH═CH—, also called ethylene), substitutedalkene (—CR═CR—, —CH═CR— and —CR═CH—), amide (—NH—CO— and —NR—CO— or—CO—NH— and —CO—NR—), azo (—N═N—), esters and thioesters (—CO—O—,—O—CO—, —CS—O— and —O—CS—) and other conjugated bonds such as (—CH═N—,—CR═N—, —N═CH— and —N═CR—), (—SiH═SiH—, —SiR═SiH—, —SiR═SiH—, and—SiR═SiR—), (—SiH═CH—, —SiR═CH—, —SiH═CR—, —SiR═CR—, —CH═SiH—, —CR═SiH—,—CH═SiR—, and —CR═SiR—). Particularly preferred B-D bonds are acetylene,alkene, amide, and substituted derivatives of these three, and azo.Especially preferred B-D bonds are acetylene, alkene and amide. Theoligomer components attached to double bonds may be in the trans or cisconformation, or mixtures. Thus, either B or D may include carbon,nitrogen or silicon. The substitution groups are as defined as above forR.

When g=0 in the Structure 1 conductive oligomer, e is preferably 1 andthe D moiety may be carbonyl or a heteroatom moiety as defined above.

As above for the Y rings, within any single conductive oligomer, the B-Dbonds (or D moieties, when g=0) may be all the same, or at least one maybe different. For example, when m is zero, the terminal B-D bond may bean amide bond, and the rest of the B-D bonds may be acetylene bonds.Generally, when amide bonds are present, as few amide bonds as possibleare preferable, but in some embodiments all the B-D bonds are amidebonds. Thus, as outlined above for the Y rings, one type of B-D bond maybe present in the conductive oligomer within a monolayer as describedbelow, and another type above the monolayer level, for example to givegreater flexibility for nucleic acid hybridization when the nucleic acidis attached via a conductive oligomer.

In the structures depicted herein, n is an integer from 1 to 50,although longer oligomers may also be used (see for example Schumm etal., Angew. Chem. Int. Ed. Engl. 1994 33(13):1360). Without being boundby theory, it appears that for efficient hybridization of nucleic acidson a surface, the hybridization should occur at a distance from thesurface, i.e. the kinetics of hybridization increase as a function ofthe distance from the surface, particularly for long oligonucleotides of200 to 300 basepairs. Accordingly, when a nucleic acid is attached via aconductive oligomer, as is more fully described below, the length of theconductive oligomer is such that the closest nucleotide of the nucleicacid is positioned from about 6 A to about 100 A (although distances ofup to 500 A may be used) from the electrode surface, with from about 15A to about 60 A being preferred and from about 25 A to about 60 A alsobeing preferred. Accordingly, n will depend on the size of the aromaticgroup, but generally will be from about 1 to about 20, with from about 2to about 15 being preferred and from about 3 to about 10 beingespecially preferred.

In the structures depicted herein, m is either 0 or 1. That is, when mis 0, the conductive oligomer may terminate in the B-D bond or D moiety,i.e. the D atom is attached to the nucleic acid either directly or via alinker. In some embodiments, for example when the conductive oligomer isattached to a phosphate of the ribose-phosphate backbone of a nucleicacid, there may be additional atoms, such as a linker, attached betweenthe conductive oligomer and the nucleic acid. Additionally, as outlinedbelow, the D atom may be the nitrogen atom of the amino-modified ribose.Alternatively, when m is 1, the conductive oligomer may terminate in Y,an aromatic group, i.e. the aromatic group is attached to the nucleicacid or linker.

As will be appreciated by those in the art, a large number of possibleconductive oligomers may be utilized. These include conductive oligomersfalling within the Structure 1 and Structure 8 formulas, as well asother conductive oligomers, as are generally known in the art, includingfor example, compounds comprising fused aromatic rings or Teflon®-likeoligomers, such as —(CF₂)_(n)—, —(CHF)_(n)— and —(CFR)_(n)—. See forexample, Schumm et al., Angew. Chem. Intl. Ed. EngI. 33:1361(1994);Grosshenny et al., Platinum Metals Rev. 40(1):26-35 (1996); Tour,Chem. Rev. 96:537-553 (1996); Hsung et al., Organometallics 14:4808-4815(1995; and references cited therein, all of which are expresslyincorporated by reference.

Particularly preferred conductive oligomers of this embodiment aredepicted below:

Structure 2 is Structure 1 when g is 1. Preferred embodiments ofStructure 2 include: e is zero, Y is pyrole or substituted pyrole; e iszero, Y is thiophene or substituted thiophene; e is zero, Y is furan orsubstituted furan; e is zero, Y is phenyl or substituted phenyl; e iszero, Y is pyridine or substituted pyridine; e is 1, B-D is acetyleneand Y is phenyl or substituted phenyl (see Structure 4 below). Apreferred embodiment of Structure 2 is also when e is one, depicted asStructure 3 below:

Preferred embodiments of Structure 3 are: Y is phenyl or substitutedphenyl and B-D is azo; Y is phenyl or substituted phenyl and B-D isacetylene; Y is phenyl or substituted phenyl and B-D is alkene; Y ispyridine or substituted pyridine and B-D is acetylene; Y is thiophene orsubstituted thiophene and B-D is acetylene; Y is furan or substitutedfuran and B-D is acetylene; Y is thiophene or furan (or substitutedthiophene or furan) and B-D are alternating alkene and acetylene bonds.

Most of the structures depicted herein utilize a Structure 3 conductiveoligomer. However, any Structure 3 oligomers may be substituted with anyof the other structures depicted herein, i.e. Structure 1 or 8 oligomer,or other conducting oligomer, and the use of such Structure 3 depictionis not meant to limit the scope of the invention.

Particularly preferred embodiments of Structure 3 include Structures 4,5, 6 and 7, depicted below:

Particularly preferred embodiments of Structure 4 include: n is two, mis one, and R is hydrogen; n is three, m is zero, and R is hydrogen; andthe use of R groups to increase solubility.

When the B-D bond is an amide bond, as in Structure 5, the conductiveoligomers are pseudopeptide oligomers. Although the amide bond inStructure 5 is depicted with the carbonyl to the left, i.e. —CONH—, thereverse may also be used, i.e. —NHCO—. Particularly preferredembodiments of Structure 5 include: n is two, m is one, and R ishydrogen; n is three, m is zero, and R is hydrogen (in this embodiment,the terminal nitrogen (the D atom) may be the nitrogen of theamino-modified ribose); and the use of R groups to increase solubility.

Preferred embodiments of Structure 6 include the first n is two, secondn is one, m is zero, and all R groups are hydrogen, or the use of Rgroups to increase solubility.

Preferred embodiments of Structure 7 include: the first n is three, thesecond n is from 1-3, with m being either 0 or 1, and the use of Rgroups to increase solubility.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 8:

In this embodiment, C are carbon atoms, n is an integer from 1 to 50, mis 0 or 1, J is a heteroatom selected from the group consisting ofoxygen, nitrogen, silicon, phosphorus, sulfur, carbonyl or sulfoxide,and G is a bond selected from alkane, alkene or acetylene, such thattogether with the two carbon atoms the C-G-C group is an alkene(—CH═CH—), substituted alkene (—CR═CR—) or mixtures thereof (—CH═CR— or—CR═CH—), acetylene (—C≡C—), or alkane (—CR₂—CR₂—, with R being eitherhydrogen or a substitution group as described herein). The G bond ofeach subunit may be the same or different than the G bonds of othersubunits; that is, alternating oligomers of alkene and acetylene bondscould be used, etc. However, when G is an alkane bond, the number ofalkane bonds in the oligomer should be kept to a minimum, with about sixor less sigma bonds per conductive oligomer being preferred. Alkenebonds are preferred, and are generally depicted herein, although alkaneand acetylene bonds may be substituted in any structure or embodimentdescribed herein as will be appreciated by those in the art.

In some embodiments, for example when ETMs are not present, if m=0 thenat least one of the G bonds is not an alkane bond.

In a preferred embodiment, the m of Structure 8 is zero. In aparticularly preferred embodiment, m is zero and G is an alkene bond, asis depicted in Structure 9:

The alkene oligomer of structure 9, and others depicted herein, aregenerally depicted in the preferred trans configuration, althougholigomers of cis or mixtures of trans and cis may also be used. Asabove, R groups may be added to alter the packing of the compositions onan electrode, the hydrophilicity or hydrophobicity of the oligomer, andthe flexibility, i.e. the rotational, torsional or longitudinalflexibility of the oligomer. n is as defined above.

In a preferred embodiment, R is hydrogen, although R may be also alkylgroups and polyethylene glycols or derivatives.

In an alternative embodiment, the conductive oligomer may be a mixtureof different types of oligomers, for example of structures 1 and 8.

In addition, the terminus of at least some of the conductive oligomersin the monolayer are electronically exposed. By “electronically exposed”herein is meant that upon the placement of an ETM in close proximity tothe terminus, and after initiation with the appropriate signal, a signaldependent on the presence of the ETM may be detected. The conductiveoligomers may or may not have terminal groups. Thus, in a preferredembodiment, there is no additional terminal group, and the conductiveoligomer terminates with one of the groups depicted in Structures 1 to9; for example, a B-D bond such as an acetylene bond. Alternatively, ina preferred embodiment, a terminal group is added, sometimes depictedherein as “Q”. A terminal group may be used for several reasons; forexample, to contribute to the electronic availability of the conductiveoligomer for detection of ETMs, or to alter the surface of the SAM forother reasons, for example to prevent non-specific binding. For example,there may be negatively charged groups on the terminus to form anegatively charged surface such that when the nucleic acid is DNA or RNAthe nucleic acid is repelled or prevented from lying down on thesurface, to facilitate hybridization. Preferred terminal groups include—NH₂, —OH, —COOH, and alkyl groups such as —CH₃, and (poly)alkyloxidessuch as (poly)ethylene glycol, with —OCH₂CH₂OH, —(OCH₂CH₂O)₂H,—(OCH₂CH₂O)₃H, and —(OCH₂CH₂O)₄H being preferred.

In one embodiment, it is possible to use mixtures of conductiveoligomers with different types of terminal groups. Thus, for example,some of the terminal groups may facilitate detection, and some mayprevent non-specific binding.

It will be appreciated that the monolayer may comprise differentconductive oligomer species, although preferably the different speciesare chosen such that a reasonably uniform SAM can be formed. Thus, forexample, when nucleic acids are covalently attached to the electrodeusing conductive oligomers, it is possible to have one type ofconductive oligomer used to attach the nucleic acid, and another typefunctioning to detect the ETM. Similarly, it may be desirable to havemixtures of different lengths of conductive oligomers in the monolayer,to help reduce non-specific signals. Thus, for example, preferredembodiments utilize conductive oligomers that terminate below thesurface of the rest of the monolayer, i.e. below the insulator layer, ifused, or below some fraction of the other conductive oligomers.Similarly, the use of different conductive oligomers may be done tofacilitate monolayer formation, or to make monolayers with alteredproperties.

The length of the species making up the monolayer will vary as needed.As outlined above, it appears that binding is more efficient at adistance from the surface. The species to which capture binding ligandsare attached (as outlined below, these can be either insulators orconductive oligomers) may be basically the same length as the monolayerforming species or longer than them, resulting in the nucleic acidsbeing more accessible to the solvent for hybridization. In someembodiments, the conductive oligomers to which the capture bindingligands are attached may be shorter than the monolayer.

The covalent attachment of the conductive oligomers and insulators maybe accomplished in a variety of ways, depending on the electrode and thecomposition of the insulators and conductive oligomers used. In apreferred embodiment, the attachment linkers with covalently attachedcapture binding ligands as depicted herein are covalently attached to anelectrode. Thus, one end or terminus of the attachment linker isattached to the capture binding ligand, and the other is attached to anelectrode. In some embodiments it may be desirable to have theattachment linker attached at a position other than a terminus, or evento have a branched attachment linker that is attached to an electrode atone terminus and to two or more capture binding ligands at othertermini, although this is not preferred. Similarly, the attachmentlinker may be attached at two sites to the electrode, as is generallydepicted in Structures 11-13. Generally, some type of linker is used, asdepicted below as “A” in Structure 10, where “X” is the conductiveoligomer, “I” is an insulator and the hatched surface is the electrode:

In this embodiment, A is a linker or atom. The choice of “A” will dependin part on the characteristics of the electrode. Thus, for example, Amay be a sulfur moiety when a gold electrode is used. Alternatively,when metal oxide electrodes are used, A may be a silicon (silane) moietyattached to the oxygen of the oxide (see for example Chen et al.,Langmuir 10:3332-3337 (1994); Lenhard et al., J. Electroanal. Chem.78:195-201 (1977), both of which are expressly incorporated byreference). When carbon based electrodes are used, A may be an aminomoiety (preferably a primary amine; see for example Deinhammer et al.,Langmuir 10:1306-1313 (1994)). Thus, preferred A moieties include, butare not limited to, silane moieties, sulfur moieties (including alkylsulfur moieties), and amino moieties. In a preferred embodiment, epoxidetype linkages with redox polymers such as are known in the art are notused.

Although depicted herein as a single moiety, the insulators andconductive oligomers may be attached to the electrode with more than one“A” moiety; the “A” moieties may be the same or different. Thus, forexample, when the electrode is a gold electrode, and “A” is a sulfuratom or moiety, multiple sulfur atoms may be used to attach theconductive oligomer to the electrode, such as is generally depictedbelow in Structures 11, 12 and 13. As will be appreciated by those inthe art, other such structures can be made. In Structures 11, 12 and 13,the A moiety is just a sulfur atom, but substituted sulfur moieties mayalso be used.

It should also be noted that similar to Structure 13, it may be possibleto have a conductive oligomer terminating in a single carbon atom withthree sulfur moieties attached to the electrode. Additionally, althoughnot always depicted herein, the conductive oligomers and insulators mayalso comprise a “Q” terminal group.

In a preferred embodiment, the electrode is a gold electrode, andattachment is via a sulfur linkage as is well known in the art, i.e. theA moiety is a sulfur atom or moiety. Although the exact characteristicsof the gold-sulfur attachment are not known, this linkage is consideredcovalent for the purposes of this invention. Representative structuresare depicted in Structure 14 and Structure 48. Structure 14 uses theStructure 3 conductive oligomer, although as for all the structuresdepicted herein, any of the conductive oligomers, or combinations ofconductive oligomers, may be used. Similarly, any of the conductiveoligomers or insulators may also comprise terminal groups as describedherein. Structure 14 depicts the “A” linker as comprising just a sulfuratom, although additional atoms may be present (i.e. linkers from thesulfur to the conductive oligomer or substitution groups).

Structure 48 depicts the “A” linker as comprising a cyclic disulfide towhich an insulator (I) is attached. Preferably the insulator is astandard monolayer forming species as defined herein, although as willbe appreciated by those of skill in the art, other insulators as areknown in the art and conductive oligomer may be used as well.

In a preferred embodiment, the electrode is a carbon electrode, i.e. aglassy carbon electrode, and attachment is via a nitrogen of an aminegroup. A representative structure is depicted in Structure 15. Again,additional atoms may be present, i.e. Z type linkers and/or terminalgroups.

In Structure 16, the oxygen atom is from the oxide of the metal oxideelectrode. The Si atom may also contain other atoms, i.e. be a siliconmoiety containing substitution groups. Other attachments for SAMs toother electrodes are known in the art; see for example Napier et al.,Langmuir, 1997, for attachment to indium tin oxide electrodes, and alsothe chemisorption of phosphates to an indium tin oxide electrode (talkby H. Holden Thorpe, CHI conference, May 4-5, 1998).

As will be appreciated by those in the art, the actual combinations andratios of the different species making up the monolayer can vary widely.Generally, three component systems are preferred, with the first speciescomprising a capture binding ligand containing species (i.e. a captureprobe, that can be attached to the electrode via either an insulator ora conductive oligomer, as is more fully described below). The secondspecies are the insulators, and the third species are electroconduitforming species. In this embodiment, the first species can comprise fromabout 90% to about 1%, with from about 20% to about 40% being preferred.When the capture binding ligands are nucleic acids and the target isnucleic acid as well, from about 30% to about 40% is especiallypreferred for short oligonucleotide targets and from about 10% to about20% is preferred for longer targets. The second species can comprisefrom about 1% to about 90%, with from about 20% to about 90% beingpreferred, and from about 40% to about 60% being especially preferred.The third species can comprise from about 1% to about 90%, with fromabout 20% to about 40% being preferred, and from about 15% to about 30%being especially preferred. Preferred ratios of first:second:thirdspecies are 2:2:1 for short targets, 1:3:1 for longer targets, withtotal thiol concentration in the 500 μM to 1 mM range, and 833 μM beingpreferred.

In a preferred embodiment, two component systems are used, comprisingthe first and second species. In this embodiment, the first species cancomprise from about 90% to about 1%, with from about 1% to about 40%being preferred, and from about 10% to about 40% being especiallypreferred. The second species can comprise from about 1% to about 90%,with from about 10% to about 60% being preferred, and from about 20% toabout 40% being especially preferred.

In a particularly preferred embodiment, two component systems are used,comprising the first species and wherein the second species is the AFMS.In this embodiment, the first species can comprise from about 90% toabout 1%, with from about 1% to about 40% being preferred, and fromabout 10% to about 40% being especially preferred. The second species,the AFMS, can comprise from about 1% to about 90%, with from about 10%to about 60% being preferred, and from about 20% to about 40% beingespecially preferred.

In a particularly preferred embodiment, the aqueous solution used toform the monolayer also comprises s a pH buffering component and azwitterionic hygroscopic agent. Preferably the buffer is Tris and thehygroscopic agent is betaine. Prefereable, the concentartion of thebuffer is about 1 mM to 1 M and more preferable about 10 mM to about 200mM. Also preferable, the concentration of the hygroscopic agent is fromabout 1 mM to about 1M and more preferably from about 100 mM to about800 mM.

The SAMs of the invention can be made in a variety of ways, includingdeposition out of organic solutions and deposition out of aqueoussolutions. The methods outlined herein use a gold electrode as theexample, although as will be appreciated by those in the art, othermetals and methods may be used as well. In one preferred embodiment,indium-tin-oxide (ITO) is used as the electrode.

In a preferred embodiment, a gold surface is first cleaned. A variety ofcleaning procedures may be employed, including, but not limited to,chemical cleaning or etchants (including Piranha solution (hydrogenperoxide/sulfuric acid) or aqua regia (hydrochloric acid/nitric acid),electrochemical methods, flame treatment, plasma treatment orcombinations thereof.

In a particularly preferred embodiment, the SAM is formed in only onestep and the second step is omitted.

Following cleaning, the gold substrate is exposed to the SAM species.When the electrode is ITO, the SAM species are phosphonate-containingspecies. This can also be done in a variety of ways, including, but notlimited to, solution deposition, gas phase deposition, microcontactprinting, spray deposition, deposition using neat components, etc. Apreferred embodiment utilizes a deposition solution comprising a mixtureof various SAM species in solution, generally thiol-containing species.Mixed monolayers that contain nucleic acids are usually prepared using atwo step procedure. The thiolated nucleic acid is deposited during thefirst deposition step (generally in the presence of at least one othermonolayer-forming species) and the mixed monolayer formation iscompleted during the second step in which a second thiol solution minusnucleic acid is added. The second step frequently involves mild heatingto promote monolayer reorganization.

In a preferred embodiment, the deposition solution is an organicdeposition solution. In this embodiment, a clean gold surface is placedinto a clean vial. A binding ligand deposition solution in organicsolvent is prepared in which the total thiol concentration is betweenmicromolar to saturation; preferred ranges include from about 1 μM to 10mM, with from about 400 uM to about 1.0 mM being especially preferred.In a preferred embodiment, the deposition solution contains thiolmodified DNA (i.e. nucleic acid attached to an attachment linker) andthiol diluent molecules (either conductive oligomers or insulators, withthe latter being preferred). The ratio of nucleic acid to diluent (ifpresent) is usually between 1000:1 to 1:1000, with from about 10:1 toabout 1:10 being preferred and 1:1 being especially preferred. Thepreferred solvents are tetrahydrofuran (THF), acetonitrile,dimethylforamide (DMF), ethanol, or mixtures thereof; generally anysolvent of sufficient polarity to dissolve the capture ligand can beused, as long as the solvent is devoid of functional groups that willreact with the surface. Sufficient nucleic acid deposition solution isadded to the vial so as to completely cover the electrode surface. Thegold substrate is allowed to incubate at ambient temperature or slightlyabove ambient temperature for a period of time ranging from seconds tohours, with 5-30 minutes being preferred. After the initial incubation,the deposition solution is removed and a solution of diluent moleculeonly (from about 1 μM to 10 mM, with from about 100 uM to about 1.0 mMbeing preferred) in organic solvent is added. The gold substrate isallowed to incubate at room temperature or above room temperature for aperiod of time (seconds to days, with from about 10 minutes to about 24hours being preferred). The gold sample is removed from the solution,rinsed in clean solvent and used.

In a preferred embodiment, an aqueous deposition solution is used. Asabove, a clean gold surface is placed into a clean vial. A nucleic aciddeposition solution in water is prepared in which the total thiolconcentration is between about 1 uM and 10 mM, with from about 1 μM toabout 200 uM being preferred. The aqueous solution frequently has saltpresent (up to saturation, with approximately 1M being preferred),however pure water can be used. The deposition solution contains thiolmodified nucleic acid and often a thiol diluent molecule. The ratio ofnucleic acid to diluent is usually between between 1000:1 to 1:1000,with from about 10:1 to about 1:10 being preferred and 1:1 beingespecially preferred. The nucleic acid deposition solution is added tothe vial in such a volume so as to completely cover the electrodesurface. The gold substrate is allowed to incubate at ambienttemperature or slightly above ambient temperature for 1-30 minutes with5 minutes usually being sufficient. After the initial incubation, thedeposition solution is removed and a solution of diluent molecule only(10 uM-1.0 mM) in either water or organic solvent is added. The goldsubstrate is allowed to incubate at room temperature or above roomtemperature until a complete monolayer is formed (10 minutes-24 hours).The gold sample is removed from the solution, rinsed in clean solventand used.

In a preferred embodiment, the deposition solution comprises azwitterionic compound, preferably betaine. Preferred embodiments utilizebetain and Tris-HCl buffers.

In a preferred embodiment, as outlined herein, a circuit board is usedas the substrate for the gold electrodes. Formation of the SAMs on thegold surface is generally done by first cleaning the boards, for examplein a 10% sulfuric acid solution for 30 seconds, detergent solutions,aqua regia, plasma, etc., as outlined herein. Following the sulfuricacid treatment, the boards are washed, for example via immersion in twoMilli-Q water baths for 1 minute each. The boards are then dried, forexample under a stream of nitrogen. Spotting of the deposition solutiononto the boards is done using any number of known spotting systems,generally by placing the boards on an X-Y table, preferably in ahumidity chamber. The size of the spotting drop will vary with the sizeof the electrodes on the boards and the equipment used for delivery ofthe solution; for example, for 250 μM size electrodes, a 30 nanoliterdrop is used. The volume should be sufficient to cover the electrodesurface completely. The drop is incubated at room temperature for aperiod of time (sec to overnight, with 5 minutes preferred) and then thedrop is removed by rinsing in a Milli-Q water bath. The boards are thenpreferably treated with a second deposition solution, generallycomprising insulator in organic solvent, preferably acetonitrile, byimmersion in a 45° C. bath. After 30 minutes, the boards are removed andimmersed in an acetonitrile bath for 30 seconds followed by a milli-Qwater bath for 30 seconds. The boards are dried under a stream ofnitrogen.

In a preferred embodiment, the electrode comprising the monolayerincluding conductive oligomers further comprises a capture bindingligand. By “capture binding ligand” or “capture binding species” or“capture probe” herein is meant a compound that is used to probe for thepresence of the target analyte, that will bind to the target analyte.Generally, the capture binding ligand allows the attachment of a targetanalyte to the electrode, for the purposes of detection. As is morefully outlined below, attachment of the target analyte to the captureprobe may be direct (i.e. the target analyte binds to the capturebinding ligand) or indirect (one or more capture extender ligands areused). By “covalently attached” herein is meant that two moieties areattached by at least one bond, including sigma bonds, pi bonds andcoordination bonds.

In a preferred embodiment, the binding is specific, and the bindingligand is part of a binding pair. By “specifically bind” herein is meantthat the ligand binds the analyte, with specificity sufficient todifferentiate between the analyte and other components or contaminantsof the test sample. However, as will be appreciated by those in the art,it will be possible to detect analytes using binding which is not highlyspecific; for example, the systems may use different binding ligands,for example an array of different ligands, and detection of anyparticular analyte is via its “signature” of binding to a panel ofbinding ligands, similar to the manner in which “electronic noses” work.This finds particular utility in the detection of chemical analytes. Thebinding should be sufficient to remain bound under the conditions of theassay, including wash steps to remove non-specific binding. In someembodiments, for example in the detection of certain biomolecules, thebinding constants of the analyte to the binding ligand will be at leastabout 10⁴-10⁶ M⁻¹, with at least about 10⁵ to 10⁹ M⁻¹ being preferredand at least about 10⁷-10⁹M⁻¹ being particularly preferred.

As will be appreciated by those in the art, the composition of thebinding ligand will depend on the composition of the target analyte.Binding ligands to a wide variety of analytes are known or can bereadily found using known techniques. For example, when the analyte is asingle-stranded nucleic acid, the binding ligand may be a complementarynucleic acid. Similarly, the analyte may be a nucleic acid bindingprotein and the capture binding ligand is either single-stranded ordouble stranded nucleic acid; alternatively, the binding ligand may be anucleic acid-binding protein when the analyte is a single ordouble-stranded nucleic acid. When the analyte is a protein, the bindingligands include proteins or small molecules. Preferred binding ligandproteins include peptides. For example, when the analyte is an enzyme,suitable binding ligands include substrates and inhibitors. As will beappreciated by those in the art, any two molecules that will associatemay be used, either as an analyte or as the binding ligand. Suitableanalyte/binding ligand pairs include, but are not limited to,antibodies/antigens, receptors/ligands, proteins/nucleic acid,enzymes/substrates and/or inhibitors, carbohydrates (includingglycoproteins and glycolipids)/lectins, proteins/proteins,proteins/small molecules; and carbohydrates and their binding partnersare also suitable analyte-binding ligand pairs. These may be wild-typeor derivative sequences. In a preferred embodiment, the binding ligandsare portions (particularly the extracellular portions) of cell surfacereceptors that are known to multimerize, such as the growth hormonereceptor, glucose transporters (particularly GLUT 4 receptor),transferrin receptor, epidermal growth factor receptor, low densitylipoprotein receptor, high density lipoprotein receptor, epidermalgrowth factor receptor, leptin receptor, interleukin receptors includingIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, VEGFreceptor, PDGF receptor, EPO receptor, TPO receptor, ciliaryneurotrophic factor receptor, prolactin receptor, and T-cell receptors.

The method of attachment of the capture binding ligand to the attachmentlinker will generally be done as is known in the art, and will depend onthe composition of the attachment linker and the capture binding ligand.In general, the capture binding ligands are attached to the attachmentlinker through the use of functional groups on each that can then beused for attachment. Preferred functional groups for attachment areamino groups, carboxy groups, oxo groups and thiol groups. Thesefunctional groups can then be attached, either directly or through theuse of a linker, sometimes depicted herein as “Z”. Linkers are known inthe art; for example, homo-or hetero-bifunctional linkers as are wellknown (see 1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference).Preferred Z linkers include, but are not limited to, alkyl groups(including substituted alkyl groups and alkyl groups containingheteroatom moieties), with short alkyl groups, esters, amide, amine,epoxy groups and ethylene glycol and derivatives being preferred. Z mayalso be a sulfone group, forming sulfonamide.

In this way, capture binding ligands comprising proteins, lectins,nucleic acids, small organic molecules, carbohydrates, etc. can beadded.

In a preferred embodiment, the capture binding ligand is attacheddirectly to the electrode as outlined herein, for example via anattachment linker. Alternatively, the capture binding ligand may utilizea capture extender component, such as depicted in FIG. 7G. In thisembodiment, the capture binding ligand comprises a first portion thatwill bind the target analyte and a second portion that can be used forattachment to the surface. FIGS. 7A-7R depict the use of a nucleic acidcomponent for binding to the surface, although this can be other bindingpartners as well.

A preferred embodiment utilizes proteinaceous capture binding ligands.As is known in the art, any number of techniques may be used to attach aproteinaceous capture binding ligand. “Protein” in this context includesproteins, polypeptides and peptides. A wide variety of techniques areknown to add moieties to proteins. One preferred method is outlined inU.S. Pat. No. 5,620,850, hereby incorporated by reference in itsentirety. The attachment of proteins to electrodes is known; see alsoHeller, Acc. Chem. Res. 23:128 (1990), and related work.

A preferred embodiment utilizes nucleic acids as the capture bindingligand, for example for when the target analyte is a nucleic acid or anucleic acid binding protein, or when the nucleic acid serves as anaptamer for binding a protein; see U.S. Pat. Nos. 5,270,163, 5,475,096,5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337, and relatedpatents, hereby incorporated by reference. In this embodiment, thenucleic acid capture binding ligand is covalently attached to theelectrode, via an “attachment linker” that can be either a conductiveoligomer or via an insulator. Thus, one end of the attachment linker isattached to a nucleic acid, and the other end (although as will beappreciated by those in the art, it need not be the exact terminus foreither) is attached to the electrode. Thus, any of structures 1-16 mayfurther comprise a nucleic acid effectively as a terminal group. Thus,the present invention provides compositions comprising binding ligandscovalently attached to electrodes as is generally depicted below inStructure 17 for a nucleic acid:

In Structure 17, the hatched marks on the left represent an electrode. Xis a conductive oligomer and I is an insulator as defined herein. F₁ isa linkage that allows the covalent attachment of the electrode and theconductive oligomer or insulator, including bonds, atoms or linkers suchas is described herein, for example as “A”, defined below. F₂ is alinkage that allows the covalent attachment of the conductive oligomeror insulator to the binding ligand, a nucleic acid in Structure 17, andmay be a bond, an atom or a linkage as is herein described. F₂ may bepart of the conductive oligomer, part of the insulator, part of thebinding ligand, or exogeneous to both, for example, as defined hereinfor “Z”.

In general, the methods, synthetic schemes and compositions useful forthe attachment of capture binding ligands, particularly nucleic acids,are outlined in WO98/20162, PCT US98/12430, PCT US98/12082; PCTUS99/01705 and PCT US99/01703, all of which are expressly incorporatedherein by reference in their entirety.

In a preferred embodiment, the capture binding ligand is covalentlyattached to the electrode via a conductive oligomer. The covalentattachment of the binding ligand and the conductive oligomer may beaccomplished in several ways, as will be appreciated by those in theart.

In a preferred embodiment, the capture binding ligand is a nucleic acid,and the attachment is via attachment to the base of the nucleoside, viaattachment to the backbone of the nucleic acid (either the ribose, thephosphate, or to an analogous group of a nucleic acid analog backbone),or via a transition metal ligand, as described below. The techniquesoutlined below are generally described for naturally occuring nucleicacids, although as will be appreciated by those in the art, similartechniques may be used with nucleic acid analogs.

In a preferred embodiment, the conductive oligomer is attached to thebase of a nucleoside of the nucleic acid. This may be done in severalways, depending on the oligomer, as is described below. In oneembodiment, the oligomer is attached to a terminal nucleoside, i.e.either the 3′ or 5′ nucleoside of the nucleic acid. Alternatively, theconductive oligomer is attached to an internal nucleoside.

The point of attachment to the basewill vary with the base. Generally,attachment at any position is possible. In some embodiments, for examplewhen the probe containing the ETMs may be used for hybridization, it ispreferred to attach at positions not involved in hydrogen bonding to thecomplementary base. Thus, for example, generally attachment is to the 5or 6 position of pyrimidines such as uridine, cytosine and thymine. Forpurines such as adenine and guanine, the linkage is preferably via the 8position. Attachment to non-standard bases is preferably done at thecomparable positions.

In one embodiment, the attachment is direct; that is, there are nointervening atoms between the conductive oligomer and the base. In thisembodiment, for example, conductive oligomers with terminal acetylenebonds are attached directly to the base. Structure 18 is an example ofthis linkage, using a Structure 3 conductive oligomer and uridine as thebase, although other bases and conductive oligomers can be used as willbe appreciated by those in the art:

It should be noted that the pentose structures depicted herein may havehydrogen, hydroxy, phosphates or other groups such as amino groupsattached. In addition, the pentose and nucleoside structures depictedherein are depicted non-conventionally, as mirror images of the normalrendering. In addition, the pentose and nucleoside structures may alsocontain additional groups, such as protecting groups, at any position,for example as needed during synthesis.

In addition, the base may contain additional modifications as needed,i.e. the carbonyl or amine groups may be altered or protected.

In an alternative embodiment, the attachment is any number of differentZ linkers, including amide and amine linkages, as is generally depictedin Structure 19 using uridine as the base and a Structure 3 oligomer:

In this embodiment, Z is a linker. Preferably, Z is a short linker ofabout 1 to about 10 atoms, with from 1 to 5 atoms being preferred, thatmay or may not contain alkene, alkynyl, amine, amide, azo, imine, etc.,bonds. Linkers are known in the art; for example, homo-orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference). Preferred Z linkers include, but arenot limited to, alkyl groups (including substituted alkyl groups andalkyl groups containing heteroatom moieties), with short alkyl groups,esters, amide, amine, epoxy groups and ethylene glycol and derivativesbeing preferred, with propyl, acetylene, and C₂ alkene being especiallypreferred. Z may also be a sulfone group, forming sulfonamide linkagesas discussed below.

In a preferred embodiment, the attachment of the nucleic acid and theconductive oligomer is done via attachment to the backbone of thenucleic acid. This may be done in a number of ways, including attachmentto a ribose of the ribose-phosphate backbone, or to the phosphate of thebackbone, or other groups of analogous backbones.

As a preliminary matter, it should be understood that the site ofattachment in this embodiment may be to a 3′ or 5′ terminal nucleotide,or to an internal nucleotide, as is more fully described below.

In a preferred embodiment, the conductive oligomer is attached to theribose of the ribose-phosphate backbone. This may be done in severalways. As is known in the art, nucleosides that are modified at eitherthe 2′ or 3′ position of the ribose with amino groups, sulfur groups,silicone groups, phosphorus groups, or oxo groups can be made (Imazawaet al., J. Org. Chem., 44:2039 (1979); Hobbs et al., J. Org. Chem.42(4):714 (1977); Verheyden et al., J. Orrg. Chem. 36(2):250 (1971);McGee et al., J. Org. Chem. 61:781-785 (1996); Mikhailopulo et al.,Liebigs. Ann. Chem. 513-519 (1993); McGee et al., Nucleosides &Nucleotides 14(6):1329 (1995), all of which are incorporated byreference). These modified nucleosides are then used to add theconductive oligomers.

A preferred embodiment utilizes amino-modified nucleosides. Theseamino-modified riboses can then be used to form either amide or aminelinkages to the conductive oligomers. In a preferred embodiment, theamino group is attached directly to the ribose, although as will beappreciated by those in the art, short linkers such as those describedherein for “Z” may be present between the amino group and the ribose.

In a preferred embodiment, an amide linkage is used for attachment tothe ribose. Preferably, if the conductive oligomer of Structures 1-3 isused, m is zero and thus the conductive oligomer terminates in the amidebond. In this embodiment, the nitrogen of the amino group of theamino-modified ribose is the “D” atom of the conductive oligomer. Thus,a preferred attachment of this embodiment is depicted in Structure 20(using the Structure 3 conductive oligomer):

As will be appreciated by those in the art, Structure 20 has theterminal bond fixed as an amide bond.

In a preferred embodiment, a heteroatom linkage is used, i.e. oxo,amine, sulfur, etc. A preferred embodiment utilizes an amine linkage.Again, as outlined above for the amide linkages, for amine linkages, thenitrogen of the amino-modified ribose may be the “D” atom of theconductive oligomer when the Structure 3 conductive oligomer is used.Thus, for example, Structures 21 and 22 depict nucleosides with theStructures 3 and 9 conductive oligomers, respectively, using thenitrogen as the heteroatom, athough other heteroatoms can be used:

In Structure 21, preferably both m and t are not zero. A preferred Zhere is a methylene group, or other aliphatic alkyl linkers. One, two orthree carbons in this position are particularly useful for syntheticreasons.

In Structure 22, Z is as defined above. Suitable linkers includemethylene and ethylene.

In an alternative embodiment, the conductive oligomer is covalentlyattached to the nucleic acid via the phosphate of the ribose-phosphatebackbone (or analog) of a nucleic acid. In this embodiment, theattachment is direct, utilizes a linker or via an amide bond. Structure23 depicts a direct linkage, and Structure 24 depicts linkage via anamide bond (both utilize the Structure 3 conductive oligomer, althoughStructure 8 conductive oligomers are also possible). Structures 23 and24 depict the conductive oligomer in the 3′ position, although the 5′position is also possible. Furthermore, both Structures 23 and 24 depictnaturally occurring phosphodiester bonds, although as those in the artwill appreciate, non-standard analogs of phosphodiester bonds may alsobe used.

In Structure 23, if the terminal Y is present (i.e. m=1), thenpreferably Z is not present (i.e. t=0). If the terminal Y is notpresent, then Z is preferably present.

Structure 24 depicts a preferred embodiment, wherein the terminal B-Dbond is an amide bond, the terminal Y is not present, and Z is a linker,as defined herein.

In a preferred embodiment, the conductive oligomer is covalentlyattached to the nucleic acid via a transition metal ligand. In thisembodiment, the conductive oligomer is covalently attached to a ligandwhich provides one or more of the coordination atoms for a transitionmetal. In one embodiment, the ligand to which the conductive oligomer isattached also has the nucleic acid attached, as is generally depictedbelow in Structure 25. Alternatively, the conductive oligomer isattached to one ligand, and the nucleic acid is attached to anotherligand, as is generally depicted below in Structure 26. Thus, in thepresence of the transition metal, the conductive oligomer is covalentlyattached to the nucleic acid. Both of these structures depict Structure3 conductive oligomers, although other oligomers may be utilized.Structures 25 and 26 depict two representative structures for nucleicacids; as will be appreciated by those in the art, it is possible toconnect other types of capture binding ligands, for exampleproteinaceous binding ligands, in a similar manner:

In the structures depicted herein, M is a metal atom, with transitionmetals being preferred. Suitable transition metals for use in theinvention include, but are not limited to, cadmium (Cd), copper (Cu),cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru),rhodium (Rh), osmium (Os), rhenium (Re), platinium (Pt), scandium (Sc),titanium (Ti), Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni),Molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir). Thatis, the first series of transition metals, the platinum metals (Ru, Rh,Pd, Os, Ir and Pt), along with Fe, Re, W, Mo and Tc, are preferred.Particularly preferred are ruthenium, rhenium, osmium, platinium, cobaltand iron.

L are the co-ligands, that provide the coordination atoms for thebinding of the metal ion. As will be appreciated by those in the art,the number and nature of the co-ligands will depend on the coordinationnumber of the metal ion. Mono-, di- or polydentate co-ligands may beused at any position. Thus, for example, when the metal has acoordination number of six, the L from the terminus of the conductiveoligomer, the L contributed from the nucleic acid, and r, add up to six.Thus, when the metal has a coordination number of six, r may range fromzero (when all coordination atoms are provided by the other two ligands)to four, when all the co-ligands are monodentate. Thus generally, r willbe from 0 to 8, depending on the coordination number of the metal ionand the choice of the other ligands.

In one embodiment, the metal ion has a coordination number of six andboth the ligand attached to the conductive oligomer and the ligandattached to the nucleic acid are at least bidentate; that is, r ispreferably zero, one (i.e. the remaining co-ligand is bidentate) or two(two monodentate co-ligands are used).

As will be appreciated in the art, the co-ligands can be the same ordifferent. Suitable ligands fall into two categories: ligands which usenitrogen, oxygen, sulfur, carbon or phosphorus atoms (depending on themetal ion) as the coordination atoms (generally referred to in theliterature as sigma (a) donors) and organometallic ligands such asmetallocene ligands (generally referred to in the literature as pi (n)donors, and depicted herein as L_(m)). Suitable nitrogen donatingligands are well known in the art and include, but are not limited to,NH₂; NHR; NRR′; pyridine; pyrazine; isonicotinamide; imidazole;bipyridine and substituted derivatives of bipyridine; terpyridine andsubstituted derivatives; phenanthrolines, particularly1,10-phenanthroline (abbreviated phen) and substituted derivatives ofphenanthrolines such as 4,7-dimethylphenanthroline anddipyridol[3,2-a:2′,3′-c]phenazine (abbreviated dppz); dipyridophenazine;1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);9,10-phenanthrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap);1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam), EDTA, EGTA andisocyanide. Substituted derivatives, including fused derivatives, mayalso be used. In some embodiments, porphyrins and substitutedderivatives of the porphyrin family may be used. See for example,Comprehensive Coordination Chemistry, Ed. Wilkinson et al., PergammonPress, 1987, Chapters 13.2 (pp73-98), 21.1 (pp. 813-898) and 21.3 (pp915-957), all of which are hereby expressly incorporated by reference.

Suitable sigma donating ligands using carbon, oxygen, sulfur andphosphorus are known in the art. For example, suitable sigma carbondonors are found in Cotton and Wilkenson, Advanced Organic Chemistry,5th Edition, John Wiley & Sons, 1988, hereby incorporated by reference;see page 38, for example. Similarly, suitable oxygen ligands includecrown ethers, water and others known in the art. Phosphines andsubstituted phosphines are also suitable; see page 38 of Cotton andWilkenson.

The oxygen, sulfur, phosphorus and nitrogen-donating ligands areattached in such a manner as to allow the heteroatoms to serve ascoordination atoms.

In a preferred embodiment, organometallic ligands are used. In additionto purely organic compounds for use as redox moieties, and varioustransition metal coordination complexes with 6-bonded organic ligandwith donor atoms as heterocyclic or exocyclic substituents, there isavailable a wide variety of transition metal organometallic compoundswith n-bonded organic ligands (see Advanced Inorganic Chemistry, 5thEd., Cotton & Wilkinson, John Wiley & Sons, 1988, chapter 26;Organometallics, A Concise Introduction, Elschenbroich et al., 2nd Ed.,1992, VCH; and Comprehensive Organometallic Chemistry II, A Review ofthe Literature 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10 &11, Pergamon Press, hereby expressly incorporated by reference). Suchorganometallic ligands include cyclic aromatic compounds such as thecyclopentadienide ion [C₅H₅(−1)] and various ring substituted and ringfused derivatives, such as the indenylide (−1) ion, that yield a classof bis(cyclopentadieyl) metal compounds, (i.e. the metallocenes); seefor example Robins et al., J. Am. Chem. Soc. 104:1882-1893 (1982); andGassman et al., J. Am. Chem. Soc. 108:4228-4229 (1986), incorporated byreference. Of these, ferrocene [(C₅H₅)₂Fe] and its derivatives areprototypical examples which have been used in a wide variety of chemical(Connelly et al., Chem. Rev. 96:877-910 (1996), incorporated byreference) and electrochemical (Geiger et al., Advances inOrganometallic Chemistry 23:1-93; and Geiger et al., Advances inOrganometallic Chemistry 24:87, incorporated by reference) electrontransfer or “redox” reactions. Metallocene derivatives of a variety ofthe first, second and third row transition metals are potentialcandidates as redox moieties that are covalently attached to either theribose ring or the nucleoside base of nucleic acid. Other potentiallysuitable organometallic ligands include cyclic arenes such as benzene,to yield bis(arene)metal compounds and their ring substituted and ringfused derivatives, of which bis(benzene)chromium is a prototypicalexample, Other acyclic n-bonded ligands such as the allyl(−1) ion, orbutadiene yield potentially suitable organometallic compounds, and allsuch ligands, in conjuction with other n-bonded and 6-bonded ligandsconstitute the general class of organometallic compounds in which thereis a metal to carbon bond. Electrochemical studies of various dimers andoligomers of such compounds with bridging organic ligands, andadditional non-bridging ligands, as well as with and without metal-metalbonds are potential candidate redox moieties in nucleic acid analysis.

When one or more of the co-ligands is an organometallic ligand, theligand is generally attached via one of the carbon atoms of theorganometallic ligand, although attachment may be via other atoms forheterocyclic ligands. Preferred organometallic ligands includemetallocene ligands, including substituted derivatives and themetalloceneophanes (see page 1174 of Cotton and Wilkenson, supra). Forexample, derivatives of metallocene ligands such asmethylcyclopentadienyl, with multiple methyl groups being preferred,such as pentamethylcyclopentadienyl, can be used to increase thestability of the metallocene. In a preferred embodiment, only one of thetwo metallocene ligands of a metallocene are denvatized.

As described herein, any combination of ligands may be used. Preferredcombinations include: a) all ligands are nitrogen donating ligands; b)all ligands are organometallic ligands; and c) the ligand at theterminus of the conductive oligomer is a metallocene ligand and theligand provided by the nucleic acid is a nitrogen donating ligand, withthe other ligands, if needed, are either nitrogen donating ligands ormetallocene ligands, or a mixture. These combinations are depicted inrepresentative structures using the conductive oligomer of Structure 3are depicted in Structures 27 (using phenanthroline and amino asrepresentative ligands), 28 (using ferrocene as the metal-ligandcombination) and 29 (using cyclopentadienyl and amino as representativeligands).

In a preferred embodiment, the ligands used in the invention showaltered fluorescent properties depending on the redox state of thechelated metal ion. As described below, this thus serves as anadditional mode of detection of electron transfer between the ETM andthe electrode.

In a preferred embodiment, as is described more fully below, the ligandattached to the nucleic acid is an amino group attached to the 2′ or 3′position of a ribose of the ribose-phosphate backbone. This ligand maycontain a multiplicity of amino groups so as to form a polydentateligand which binds the metal ion. Other preferred ligands includecyclopentadiene and phenanthroline.

The use of metal ions to connect the nucleic acids can serve as aninternal control or calibration of the system, to evaluate the number ofavailable nucleic acids on the surface. However, as will be appreciatedby those in the art, if metal ions are used to connect the nucleic acidsto the conductive oligomers, it is generally desirable to have thismetal ion complex have a different redox potential than that of the ETMsused in the rest of the system, as described below. This is generallytrue so as to be able to distinguish the presence of the capture probefrom the presence of the target sequence. This may be useful foridentification, calibration and/or quantification. Thus, the amount ofcapture probe on an electrode may be compared to the amount ofhybridized double stranded nucleic acid to quantify the amount of targetsequence in a sample. This is quite significant to serve as an internalcontrol of the sensor or system. This allows a measurement either priorto the addition of target or after, on the same molecules that will beused for detection, rather than rely on a similar but different controlsystem. Thus, the actual molecules that will be used for the detectioncan be quantified prior to any experiment. This is a significantadvantage over prior methods.

In a preferred embodiment, the capture probe nucleic acids arecovalently attached to the electrode via an insulator. The attachment ofnucleic acids to insulators such as alkyl groups is well known, and canbe done to the base or the backbone, including the ribose or phosphatefor backbones containing these moieties, or to alternate backbones fornucleic acid analogs.

In a preferred embodiment, the capture binding ligands are covalentlyattached to the electrode via an insulator. The attachment of a varietyof binding ligands such as proteins and nucleic acids to insulators suchas alkyl groups is well known, and can be done to the nucleic acid basesor the backbone, including the ribose or phosphate for backbonescontaining these moieties, or to alternate backbones for nucleic acidanalogs, or to the side chains or backbone of the amino acids.

In a preferred embodiment, there may be one or more different capturebinding ligand species (sometimes referred to herein as “anchorligands”, “anchor probes” or “capture probes” with the phrase “probe”generally referring to nucleic acid species) on the surface, as isgenerally depicted in the Figures. In some embodiments, there may be onetype of capture binding ligand, or one type of capture binding ligandextender, as is more fully described below. Alternatively, differentcapture binding ligands, or one capture binding ligand with amultiplicity of different capture extender binding ligands can be used.Similarly, when nucleic acid systems are used, it may be desirable touse auxiliary capture probes that comprise relatively short probesequences, that can be used to “tack down” components of the system, forexample the recruitment linkers, to increase the concentration of ETMsat the surface.

Thus the present invention provides electrodes comprising monolayerscomprising conductive oligomers and capture binding ligands, useful intarget analyte detection systems.

In a preferred embodiment, the compositions further comprise a solutionbinding ligand. Solution binding ligands are similar to capture bindingligands, in that they bind to target analytes. The solution bindingligand may be the same or different from the capture binding ligand.Generally, the solution binding ligands are not directly attached to thesurface, although as depicted in FIG. 5A they may be. The solutionbinding ligand either directly comprises a recruitment linker thatcomprises at least one ETM, or the recruitment linker is part of a labelprobe that will bind to the solution binding ligand.

Thus, “recruitment linkers” or “signal carriers” with covalentlyattached ETMs are provided. The terms “electron donor moiety”, “electronacceptor moiety”, and “ETMs” (ETMs) or grammatical equivalents hereinrefers to molecules capable of electron transfer under certainconditions. It is to be understood that electron donor and acceptorcapabilities are relative; that is, a molecule which can lose anelectron under certain experimental conditions will be able to accept anelectron under different experimental conditions. It is to be understoodthat the number of possible electron donor moieties and electronacceptor moieties is very large, and that one skilled in the art ofelectron transfer compounds will be able to utilize a number ofcompounds in the present invention. Preferred ETMs include, but are notlimited to, transition metal complexes, organic ETMs, and electrodes.

In a preferred embodiment, the ETMs are transition metal complexes.Transition metals are those whose atoms have a partial or complete dshell of electrons. Suitable transition metals for use in the inventionare listed above.

The transition metals are complexed with a variety of ligands, L,defined above, to form suitable transition metal complexes, as is wellknown in the art.

In addition to transition metal complexes, other organic electron donorsand acceptors may be covalently attached to the nucleic acid for use inthe invention. These organic molecules include, but are not limited to,riboflavin, xanthene dyes, azine dyes, acridine orange,N,/N′-dimethyl-2,7-diazapyrenium dichloride (DAp²⁺), methylviologen,ethidium bromide, quinones such asN,N′-dimethylanthra(2,1,9-def:6,5,10-d′e′f′)diisoquinoline dichloride(ADIQ²⁺); porphyrins ([meso-tetrakis(N-methyl-x-pyridinium)porphyrintetrachloride], varlamine blue B hydrochloride, Bindschedler's green;2,6-dichloroindophenol, 2,6-dibromophenolindophenol; Brilliant crestblue (3-amino-9-dimethyl-amino-10-methylphenoxyazine chloride),methylene blue; Nile blue A (aminoaphthodiethylaminophenoxazinesulfate), indigo-5,5′,7,7′-tetrasulfonic acid, indigo-5,5′,7-trisulfonicacid; phenosafranine, indigo-5-monosulfonic acid; safranine T;bis(dimethylglyoximato)-iron(II) chloride; induline scarlet, neutralred, anthracene, coronene, pyrene, 9-phenylanthracene, rubrene,binaphthyl, DPA, phenothiazene, fluoranthene, phenanthrene, chrysene,1,8-diphenyl-1,3,5,7-octatetracene, naphthalene, acenaphthalene,perylene, TMPD and analogs and subsitituted derivatives of thesecompounds.

In one embodiment, the electron donors and acceptors are redox proteinsas are known in the art. However, redox proteins in many embodiments arenot preferred.

The choice of the specific ETMs will be influenced by the type ofelectron transfer detection used, as is generally outlined below.Preferred ETMs are metallocenes, with ferrocene being particularlypreferred.

In a preferred embodiment, a plurality of ETMs are used. As is shown inthe examples, the use of multiple ETMs provides signal amplification andthus allows more sensitive detection limits. Accordingly, pluralities ofETMs are preferred, with at least about 2 ETMs per recruitment linkerbeing preferred, and at least about 10 being particularly preferred, andat least about 20 to 50 being especially preferred. In some instances,very large numbers of ETMs (100 to 1000) can be used.

As will be appreciated by those in the art, the portion of the labelprobe (or target, in some embodiments) that comprises the ETMs (termedherein a “recruitment linker” or “signal carrier”) can be nucleic acid,or it can be a non-nucleic acid linker that links the solution bindingligand to the ETMs. Thus, as will be appreciated by those in the art,there are a variety of configurations that can be used. In a preferredembodiment, the recruitment linker is nucleic acid (including analogs),and attachment of the ETMs can be via (1) a base; (2) the backbone,including the ribose, the phosphate, or comparable structures in nucleicacid analogs; (3) nucleoside replacement, described below; or (4)metallocene polymers, as described below. In a preferred embodiment, therecruitment linker is non-nucleic acid, and can be either a metallocenepolymer or an alkyl-type polymer (including heteroalkyl, as is morefully described below) containing ETM substitution groups.

In a preferred embodiment, the recruitment linker is a nucleic acid, andcomprises covalently attached ETMs. The ETMs may be attached tonucleosides within the nucleic acid in a variety of positions. Preferredembodiments include, but are not limited to, (1) attachment to the baseof the nucleoside, (2) attachment of the ETM as a base replacement, (3)attachment to the backbone of the nucleic acid, including either to aribose of the ribose-phosphate backbone or to a phosphate moiety, or toanalogous structures in nucleic acid analogs, and (4) attachment viametallocene polymers, with the latter being preferred.

In addition, as is described below, when the recruitment linker isnucleic acid, it may be desirable to use secondary label probes, thathave a first portion that will hybridize to a portion of the primarylabel probes and a second portion comprising a recruitment linker as isdefined herein. This is generally depicted in FIGS. 7Q and 7R; this issimilar to the use of an amplifier probe, except that both the primaryand the secondary label probes comprise ETMs.

In a preferred embodiment, the ETM is attached to the base of anucleoside as is generally outlined above for attachment of theconductive oligomer. Attachment can be to an internal nucleoside or aterminal nucleoside.

The covalent attachment to the base will depend in part on the ETMchosen, but in general is similar to the attachment of conductiveoligomers to bases, as outlined above. Attachment may generally be doneto any position of the base. In a preferred embodiment, the ETM is atransition metal complex, and thus attachment of a suitable metal ligandto the base leads to the covalent attachment of the ETM. Alternatively,similar types of linkages may be used for the attachment of organicETMs, as will be appreciated by those in the art.

In one embodiment, the C4 attached amino group of cytosine, the C6attached amino group of adenine, or the C2 attached amino group ofguanine may be used as a transition metal ligand.

Ligands containing aromatic groups can be attached via acetylenelinkages as is known in the art (see Comprehensive Organic Synthesis,Trost et al., Ed., Pergamon Press, Chapter 2.4: Coupling ReactionsBetween sp² and sp Carbon Centers, Sonogashira, pp521-549, andpp950-953, hereby incorporated by reference). Structure 30 depicts arepresentative structure in the presence of the metal ion and any othernecessary ligands; Structure 30 depicts uridine, although as for all thestructures herein, any other base may also be used.

L_(a) is a ligand, which may include nitrogen, oxygen, sulfur orphosphorus donating ligands or organometallic ligands such asmetallocene ligands. Suitable L_(a) ligands include, but not limited to,phenanthroline, imidazole, bpy and terpy. L_(r) and M are as definedabove. Again, it will be appreciated by those in the art, a linker (“Z”)may be included between the nucleoside and the ETM.

Similarly, as for the conductive oligomers, the linkage may be doneusing a linker, which may utilize an amide linkage (see generally Telseret al., J. Am. Chem. Soc. 111:7221-7226 (1989); Telser et al., J. Am.Chem. Soc. 111:7226-7232 (1989), both of which are expresslyincorporated by reference). These structures are generally depictedbelow in Structure 31, which again uses uridine as the base, although asabove, the other bases may also be used:

In this embodiment, L is a ligand as defined above, with Lr and M asdefined above as well. Preferably, L is amino, phen, byp and terpy.

In a preferred embodiment, the ETM attached to a nucleoside is ametallocene; i.e. the L and L_(r) of Structure 31 are both metalloceneligands, L_(m), as described above. Structure 32 depicts a preferredembodiment wherein the metallocene is ferrocene, and the base isuridine, although other bases may be used:

Preliminary data suggest that Structure 32 may cyclize, with the secondacetylene carbon atom attacking the carbonyl oxygen, forming afuran-like structure. Preferred metallocenes include ferrocene,cobaltocene and osmiumocene.

In a preferred embodiment, the ETM is attached to a ribose at anyposition of the ribose-phosphate backbone of the nucleic acid, i.e.either the 5′ or 3′ terminus or any internal nucleoside. Ribose in thiscase can include ribose analogs. As is known in the art, nucleosidesthat are modified at either the 2′ or 3′ position of the ribose can bemade, with nitrogen, oxygen, sulfur and phosphorus-containingmodifications possible. Amino-modified and oxygen-modified ribose ispreferred. See generally PCT publication WO 95/15971, incorporatedherein by reference. These modification groups may be used as atransition metal ligand, or as a chemically functional moiety forattachment of other transition metal ligands and organometallic ligands,or organic electron donor moieties as will be appreciated by those inthe art. In this embodiment, a linker such as depicted herein for “Z”may be used as well, or a conductive oligomer between the ribose and theETM. Preferred embodiments utilize attachment at the 2′ or 3′ positionof the ribose, with the 2′ position being preferred. Thus for example,the conductive oligomers depicted in Structure 13, 14 and 15 may bereplaced by ETMs; alternatively, the ETMs may be added to the freeterminus of the conductive oligomer.

In a preferred embodiment, a metallocene serves as the ETM, and isattached via an amide bond as depicted below in Structure 33. Theexamples outline the synthesis of a preferred compound when themetallocene is ferrocene.

In a preferred embodiment, amine linkages are used, as is generallydepicted in Structure 34.

Z is a linker, as defined herein, with 1-16 atoms being preferred, and2-4 atoms being particularly preferred, and t is either one or zero.

In a preferred embodiment, oxo linkages are used, as is generallydepicted in Structure 35.

In Structure 35, Z is a linker, as defined herein, and t is either oneor zero. Preferred Z linkers include alkyl groups including heteroalkylgroups such as (CH₂)_(n) and (CH₂CH₂O)n, with n from 1 to 10 beingpreferred, and n=1 to 4 being especially preferred, and n=4 beingparticularly preferred.

Linkages utilizing other heteroatoms are also possible.

In a preferred embodiment, an ETM is attached to a phosphate at anyposition of the ribose-phosphate backbone of the nucleic acid. This maybe done in a variety of ways. In one embodiment, phosphodiester bondanalogs such as phosphoramide or phosphoramidite linkages may beincorporated into a nucleic acid, where the heteroatom (i.e. nitrogen)serves as a transition metal ligand (see PCT publication WO 95/15971,incorporated by reference). Alternatively, the conductive oligomersdepicted in Structures 23 and 24 may be replaced by ETMs. In a preferredembodiment, the composition has the structure shown in Structure 36.

In Structure 361, the ETM is attached via a phosphate linkage, generallythrough the use of a linker, Z. Preferred Z linkers include alkylgroups, including heteroalkyl groups such as (CH₂)_(n), (CH₂CH₂O)_(n),with n from 1 to 10 being preferred, and n=1 to 4 being especiallypreferred, and n=4 being particularly preferred.

When the ETM is attached to the base or the backbone of the nucleoside,it is possible to attach the ETMs via “dendrimer” structures, as is morefully outlined below. Alkyl-based linkers can be used to create multiplebranching structures comprising one or more ETMs at the terminus of eachbranch. Generally, this is done by creating branch points containingmultiple hydroxy groups, which optionally can then be used to addadditional branch points. The terminal hydroxy groups can then be usedin phosphoramidite reactions to add ETMs, as is generally done below forthe nucleoside replacement and metallocene polymer reactions.

In a preferred embodiment, an ETM such as a metallocene is used as a“nucleoside replacement”, serving as an ETM. For example, the distancebetween the two cyclopentadiene rings of ferrocene is similar to theorthongonal distance between two bases in a double stranded nucleicacid. Other metallocenes in addition to ferrocene may be used, forexample, air stable metallocenes such as those containing cobalt orruthenium. Thus, metallocene moieties may be incorporated into thebackbone of a nucleic acid, as is generally depicted in Structure 37(nucleic acid with a ribose-phosphate backbone) and Structure 38(peptide nucleic acid backbone). Structures 37 and 38 depict ferrocene,although as will be appreciated by those in the art, other metallocenesmay be used as well. In general, air stable metallocenes are preferred,including metallocenes utilizing ruthenium and cobalt as the metal.

In Structure 37, Z is a linker as defined above, with generally short,alkyl groups, including heteroatoms such as oxygen being preferred.Generally, what is important is the length of the linker, such thatminimal perturbations of a double stranded nucleic acid is effected, asis more fully described below. Thus, methylene, ethylene, ethyleneglycols, propylene and butylene are all preferred, with ethylene andethylene glycol being particularly preferred. In addition, each Z linkermay be the same or different. Structure 37 depicts a ribose-phosphatebackbone, although as will be appreciated by those in the art, nucleicacid analogs may also be used, including ribose analogs and phosphatebond analogs.

In Structure 38, preferred Z groups are as listed above, and again, eachZ linker can be the same or different. As above, other nucleic acidanalogs may be used as well.

In addition, although the structures and discussion above depictsmetallocenes, and particularly ferrocene, this same general idea can beused to add ETMs in addition to metallocenes, as nucleoside replacementsor in polymer embodiments, described below. Thus, for example, when theETM is a transition metal complex other than a metallocene, comprisingone, two or three (or more) ligands, the ligands can be functionalizedas depicted for the ferrocene to allow the addition of phosphoramiditegroups. Particularly preferred in this embodiment are complexescomprising at least two ring (for example, aryl and substituted aryl)ligands, where each of the ligands comprises functional groups forattachment via phosphoramidite chemistry. As will be appreciated bythose in the art, this type of reaction, creating polymers of ETMseither as a portion of the backbone of the nucleic acid or as “sidegroups” of the nucleic acids, to allow amplification of the signalsgenerated herein, can be done with virtually any ETM that can befunctionalized to contain the correct chemical groups.

Thus, by inserting a metallocene such as ferrocene (or other ETM) intothe backbone of a nucleic acid, nucleic acid analogs are made; that is,the invention provides nucleic acids having a backbone comprising atleast one metallocene. This is distinguished from nucleic acids havingmetallocenes attached to the backbone, i.e. via a ribose, a phosphate,etc. That is, two nucleic acids each made up of a traditional nucleicacid or analog (nucleic acids in this case including a singlenucleoside), may be covalently attached to each other via a metallocene.Viewed differently, a metallocene derivative or substituted metalloceneis provided, wherein each of the two aromatic rings of the metallocenehas a nucleic acid substitutent group.

In addition, as is more fully outlined below, it is possible toincorporate more than one metallocene into the backbone, either withnucleotides in between and/or with adjacent metallocenes. When adjacentmetallocenes are added to the backbone, this is similar to the processdescribed below as “metallocene polymers”; that is, there are areas ofmetallocene polymers within the backbone.

In addition to the nucleic acid substitutent groups, it is alsodesirable in some instances to add additional substituent groups to oneor both of the aromatic rings of the metallocene (or ETM). For example,as these nucleoside replacements are generally part of probe sequencesto be hybridized with a substantially complementary nucleic acid, forexample a target sequence or another probe sequence, it is possible toadd substitutent groups to the metallocene rings to facilitate hydrogenbonding to the base or bases on the opposite strand. These may be addedto any position on the metallocene rings. Suitable substitutent groupsinclude, but are not limited to, amide groups, amine groups, carboxylicacids, and alcohols, including substituted alcohols. In addition, thesesubstitutent groups can be attached via linkers as well, although ingeneral this is not preferred.

In addition, substituent groups on an ETM, particularly metallocenessuch as ferrocene, may be added to alter the redox properties of theETM. Thus, for example, in some embodiments, as is more fully describedbelow, it may be desirable to have different ETMs attached in differentways (i.e. base or ribose attachment), on different probes, or fordifferent purposes (for example, calibration or as an internalstandard). Thus, the addition of substituent groups on the metallocenemay allow two different ETMs to be distinguished.

In order to generate these metallocene-backbone nucleic acid analogs,the intermediate components are also provided. Thus, in a preferredembodiment, the invention provides phosphoramidite metallocenes, asgenerally depicted in Structure 39:

In Structure 39, PG is a protecting group, generally suitable for use innucleic acid synthesis, with DMT, MMT and TMT all being preferred. Thearomatic rings can either be the rings of the metallocene, or aromaticrings of ligands for transition metal complexes or other organic ETMs.The aromatic rings may be the same or different, and may be substitutedas discussed herein.

Structure 40 depicts the ferrocene derivative:

These phosphoramidite analogs can be added to standard oligonucleotidesyntheses as is known in the art.

Structure 41 depicts the ferrocene peptide nucleic acid (PNA) monomer,that can be added to PNA synthesis (or regular protein synthesis) as isknown in the art and as illustrated in PCT/US99/10104 andPCT/US00/20476, incorporated herein by reference:

In Structure 41, the PG protecting group is suitable for use in peptidenucleic acid synthesis, with MMT, boc and Fmoc being preferred.

These same intermediate compounds can be used to form ETM or metallocenepolymers, which are added to the nucleic acids, rather than as backbonereplacements, as is more fully described below.

In a preferred embodiment, the ETMs are attached as polymers, forexample as metallocene polymers, in a “branched” configuration similarto the “branched DNA” embodiments herein and as outlined in U.S. Pat.No. 5,124,246, using modified functionalized nucleotides. The generalidea is as follows. A modified phosphoramidite nucleotide is generatedthat can ultimately contain a free hydroxy group that can be used in theattachment of phosphoramidite ETMs such as metallocenes. This freehydroxy group could be on the base or the backbone, such as the riboseor the phosphate (although as will be appreciated by those in the art,nucleic acid analogs containing other structures can also be used). Themodified nucleotide is incorporated into a nucleic acid, and any hydroxyprotecting groups are removed, thus leaving the free hydroxyl. Upon theaddition of a phosphoramidite ETM such as a metallocene, as describedabove in structures 39 and 40, ETMs, such as metallocene ETMs, areadded. Additional phosphoramidite ETMs such as metallocenes can beadded, to form “ETM polymers”, including “metallocene polymers” asdepicted in PCT/US99/10104. In addition, Win some embodiments, it isdesirable to increase the solubility of the polymers by adding a“capping” group to the terminal ETM in the polymer. Other suitablesolubility enhancing “capping” groups will be appreciated by those inthe art. It should be noted that these solubility enhancing groups canbe added to the polymers in other places, including to the ligand rings,for example on the metallocenes as discussed herein.

Briefly, the 2′ position of a ribose of a phosphoramidite nucleotide isfirst functionalized to contain a protected hydroxy group, in this casevia an oxo-linkage, although any number of linkers can be used, as isgenerally described herein for Z linkers. The protected modifiednucleotide is then incorporated via standard phosphoramidite chemistryinto a growing nucleic acid. The protecting group is removed, and thefree hydroxy group is used, again using standard phosphoramiditechemistry to add a phosphoramidite metallocene such as ferrocene. Asimilar reaction is possible for nucleic acid analogs. For example,using peptide nucleic acids and the metallocene monomer shown inStructure 41, peptide nucleic acid structures containing metallocenepolymers could be generated.

Thus, the present invention provides recruitment linkers of nucleicacids comprising “branches” of metallocene polymers. Preferredembodiments also utilize metallocene polymers from one to about 50metallocenes in length, with from about 5 to about 20 being preferredand from about 5 to about 10 being especially preferred.

In addition, when the recruitment linker is nucleic acid, anycombination of ETM attachments may be done.

In a preferred embodiment, the recruitment linker is not nucleic acid,and instead may be any sort of linker or polymer. As will be appreciatedby those in the art, generally any linker or polymer that can bemodified to contain ETMs can be used. In general, the polymers orlinkers should be reasonably soluble and contain suitable functionalgroups for the addition of ETMs.

As used herein, a “recruitment polymer” comprises at least two or threesubunits, which are covalently attached. At least some portion of themonomeric subunits contain functional groups for the covalent attachmentof ETMs. In some embodiments coupling moieties are used to covalentlylink the subunits with the ETMs. Preferred functional groups forattachment are amino groups, carboxy groups, oxo groups and thiolgroups, with amino groups being particularly preferred. As will beappreciated by those in the art, a wide variety of recruitment polymersare possible.

Suitable linkers include, but are not limited to, alkyl linkers(including heteroalkyl (including (poly)ethylene glycol-typestructures), substituted alkyl, aryalkyl linkers, etc. As above for thepolymers, the linkers will comprise one or more functional groups forthe attachment of ETMs, which will be done as will be appreciated bythose in the art, for example through the use homo-orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference).

Suitable recruitment polymers include, but are not limited to,functionalized styrenes, such as amino styrene, functionalized dextrans,and polyamino acids. Preferred polymers are polyamino acids (bothpoly-D-amino acids and poly-L-amino acids), such as polylysine, andpolymers containing lysine and other amino acids being particularlypreferred. Other suitable polyamino acids are polyglutamic acid,polyaspartic acid, co-polymers of lysine and glutamic or aspartic acid,co-polymers of lysine with alanine, tyrosine, phenylalanine, serine,tryptophan, and/or proline.

In a preferred embodiment, the recruitment linker comprises ametallocene polymer, as is described above.

The attachment of the recruitment linkers to either the solution bindingligand or the first portion of the label probe will depend on thecomposition of the recruitment linker and of the label and/or bindingligand, as will be appreciated by those in the art. When either thelabel probe or the binding ligand is nucleic acid, nucleic acidrecruitment linkers are generally formed during the synthesis of thefirst species, with incorporation of nucleosides containing ETMs asrequired. Alternatively, the first portion of the label probe or thebinding ligand and the recruitment linker may be made separately, andthen attached. When they are both nucleic acid, there may be anoverlapping section of complementarity, forming a section of doublestranded nucleic acid that can then be chemically crosslinked, forexample by using psoralen as is known in the art.

When non-nucleic acid recruitment linkers are used, attachment of thelinker/polymer of the recruitment linker will be done generally usingstandard chemical techniques, such as will be appreciated by those inthe art. For example, when alkyl-based linkers are used, attachment canbe similar to the attachment of insulators to nucleic acids.

In addition, it is possible to have recruitment linkers that aremixtures of nucleic acids and non-nucleic acids, either in a linear form(i.e. nucleic acid segments linked together with alkyl linkers) or inbranched forms (nucleic acids with alkyl “branches” that may containETMs and may be additionally branched).

In a preferred embodiment, it is the target sequence itself that carriesthe ETMs, rather than the recruitment linker of a label probe. Forexample, as is more fully described below, it is possible toenzymatically add triphosphate nucleotides comprising the ETMs of theinvention to a growing nucleic acid, for example during a polymerasechain reaction (PCR). As will be recognized by those in the art, whileseveral enzymes have been shown to generally tolerate modifiednucleotides, some of the modified nucleotides of the invention, forexample the “nucleoside replacement” embodiments and putatively some ofthe phosphate attachments, may or may not be recognized by the enzymesto allow incorporation into a growing nucleic acid. Therefore, preferredattachments in this embodiment are to the base or ribose of thenucleotide.

Thus, for example, PCR amplification of a target sequence, as is wellknown in the art, will result in target sequences comprising ETMs,generally randomly incorporated into the sequence.

Alternatively, as outlined more fully below, it is possible toenzymatically add nucleotides comprising ETMs to the terminus of anucleic acid, for example a target nucleic acid. In this embodiment, aneffective “recruitment linker” is added to the terminus of the targetsequence, that can then be used for detection. Thus the inventionprovides compositions utilizing electrodes comprising monolayers ofconductive oligomers and capture probes, and target sequences thatcomprises a first portion that is capable of hybridizing to a componentof an assay complex, and a second portion that does not hybridize to acomponent of an assay complex and comprises at least one covalentlyattached electron transfer moiety. Similarly, methods utilizing thesecompositions are also provided.

It is also possible to have ETMs connected to probe sequences, i.e.sequences designed to hybridize to complementary sequences. Thus, ETMsmay be added to non-recruitment linkers as well. For example, there maybe ETMs added to sections of label probes that do hybridize tocomponents of the assay complex, for example the first portion, or tothe target sequence as outlined above and depicted in FIG. 7R. TheseETMs may be used for electron transfer detection in some embodiments, orthey may not, depending on the location and system. For example, in someembodiments, when for example the target sequence containing randomlyincorporated ETMs is hybridized directly to the capture probe, as isdepicted in FIGS. 7A and 7B, there may be ETMs in the portionhybridizing to the capture probe. If the capture probe is attached tothe electrode using a conductive oligomer, these ETMs can be used todetect electron transfer as has been previously described.Alternatively, these ETMs may not be specifically detected.

Similarly, in some embodiments, when the recruitment linker is nucleicacid, it may be desirable in some instances to have some or all of therecruitment linker be double stranded. In one embodiment, there may be asecond recruitment linker, substantially complementary to the firstrecruitment linker, that can hybridize to the first recruitment linker.In a preferred embodiment, the first recruitment linker comprises thecovalently attached ETMs. In an alternative embodiment, the secondrecruitment linker contains the ETMs, and the first recruitment linkerdoes not, and the ETMs are recruited to the surface by hybridization ofthe second recruitment linker to the first. In yet another embodiment,both the first and second recruitment linkers comprise ETMs. It shouldbe noted, as discussed above, that nucleic acids comprising a largenumber of ETMs may not hybridize as well, i.e. the Tm may be decreased,depending on the site of attachment and the characteristics of the ETM.Thus, in general, when multiple ETMs are used on hybridizing strands,generally there are less than about 5, with less than about 3 beingpreferred, or alternatively the ETMs should be spaced sufficiently farapart that the intervening nucleotides can sufficiently hybridize toallow good kinetics.

In one embodiment, when nucleic acid targets and/or binding ligandsand/or recruitment linkers are used, non-covalently attached ETMs may beused. In one embodiment, the ETM is a hybridization indicator.Hybridization indicators serve as an ETM that will preferentiallyassociate with double stranded nucleic acid is added, usuallyreversibly, similar to the method of Millan et al., Anal. Chem.65:2317-2323 (1993); Millan et al., Anal. Chem. 662943-2948 (1994), bothof which are hereby expressly incorporated by reference. In thisembodiment, increases in the local concentration of ETMs, due to theassociation of the ETM hybridization indicator with double strandednucleic acid at the surface, can be monitored using the monolayerscomprising the conductive oligomers. Hybridization indicators includeintercalators and minor and/or major groove binding moieties. In apreferred embodiment, intercalators may be used; since intercalationgenerally only occurs in the presence of double stranded nucleic acid,only in the presence of double stranded nucleic acid will the ETMsconcentrate. Intercalating transition metal complex ETMs are known inthe art. Similarly, major or minor groove binding moieties, such asmethylene blue, may also be used in this embodiment.

Similarly, the systems of the invention may utilize non-covalentlyattached ETMs, as is generally described in Napier et al., Bioconj.Chem. 8:906 (1997), hereby expressly incorporated by reference. In thisembodiment, changes in the redox state of certain molecules as a resultof the presence of DNA (i.e. guanine oxidation by ruthenium complexes)can be detected using the SAMs comprising conductive oligomers as well.

Thus, the present invention provides electrodes comprising monolayerscomprising conductive oligomers, generally including capture bindingligands, and either binding ligands or label probes that will bind tothe binding ligands comprising recruitment linkers containing ETMs.

In a preferred embodiment, the compositions of the invention are used todetect target analytes in a sample. In a preferred embodiment, thetarget analyte is a nucleic acid, and thus detection of target sequencesis done. The term “target sequence” or grammatical equivalents hereinmeans a nucleic acid sequence on a single strand of nucleic acid. Thetarget sequence may be a portion of a gene, a regulatory sequence,genomic DNA, cDNA, RNA including mRNA and rRNA, or others. It may be anylength, with the understanding that longer sequences are more specific.As will be appreciated by those in the art, the complementary targetsequence may take many forms. For example, it may be contained within alarger nucleic acid sequence, i.e. all or part of a gene or mRNA, arestriction fragment of a plasmid or genomic DNA, among others. As isoutlined more fully below, probes are made to hybridize to targetsequences to determine the presence or absence of the target sequence ina sample. Generally speaking, this term will be understood by thoseskilled in the art. The target sequence may also be comprised ofdifferent target domains; for example, a first target domain of thesample target sequence may hybridize to a capture probe or a portion ofcapture extender probe, a second target domain may hybridize to aportion of an amplifier probe, a label probe, or a different capture orcapture extender probe, etc. The target domains may be adjacent orseparated. The terms “first” and “second” are not meant to confer anorientation of the sequences with respect to the 5′-3′ orientation ofthe target sequence. For example, assuming a 5′-3′ orientation of thecomplementary target sequence, the first target domain may be locatedeither 5′ to the second domain, or 3′ to the second domain.

If required, the target analyte is prepared using known techniques. Forexample, the sample may be treated to lyse the cells, using known lysisbuffers, electroporation, etc., with purification occuring as needed, aswill be appreciated by those in the art. In a preferred embodiment, whenthe target analyte is nucleic acid, amplification may be done, includingPCR and other amplification techniques as outlined in PCT US99/01705,incorporated herein by reference in its entirety. When the targetanalyte is a nucleic acid, probes of the present invention are designedto be complementary to a target sequence (either the target sequence ofthe sample or to other probe sequences, as is described below), suchthat hybridization of the target sequence and the probes of the presentinvention occurs. As outlined below, this complementarity need not beperfect; there may be any number of base pair mismatches which willinterfere with hybridization between the target sequence and the singlestranded nucleic acids of the present invention. However, if the numberof mutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, by “substantially complementary”herein is meant that the probes are sufficiently complementary to thetarget sequences to hybridize under normal reaction conditions.

Generally, the nucleic acid compositions of the invention are useful asoligonucleotide probes. As is appreciated by those in the art, thelength of the probe will vary with the length of the target sequence andthe hybridization and wash conditions. Generally, oligonucleotide probesrange from about 8 to about 50 nucleotides, with from about 10 to about30 being preferred and from about 12 to about 25 being especiallypreferred. In some cases, very long probes may be used, e.g. 50 to200-300 nucleotides in length. Thus, in the structures depicted herein,nucleosides may be replaced with nucleic acids.

A variety of hybridization conditions may be used in the presentinvention, including high, moderate and low stringency conditions; seefor example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2dEdition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, etal, hereby incorporated by reference. The hybridization conditions mayalso vary when a non-ionic backbone, i.e. PNA is used, as is known inthe art. In addition, cross-linking agents may be added after targetbinding to cross-link, i.e. covalently attach, the two strands of thehybridization complex.

When gold electrodes are used, a C6 insulator, comprising6-mercaptohexanol, is included in the hybridization buffer.

As will be appreciated by those in the art, the nucleic acid systems ofthe invention may take on a large number of different configurations, asis generally depicted in the figures. In general, there are three typesof systems that can be used: (1) systems in which the target analyteitself is labeled with ETMs (i.e. the use of a target analyte analog,for non-nucleic acid systems, or, for nucleic acid systems, the targetsequence is labeled); (2) systems in which label probes (or capturebinding ligands with recruitment linkers) directly bind (i.e. hybridizefor nucleic acids) to the target analytes; and (3) systems in whichlabel probes comprising recruitment linkers are indirectly bound to thetarget analytes, for example through the use of amplifier probes.

In all three of these systems, it is preferred, although not required,that the target analyte be immobilized on the electrode surface. This ispreferably done using capture binding ligands and optionally one or morecapture extender ligands. When only capture binding ligands areutilized, it is necessary to have unique capture binding ligands foreach target analyte; that is, the surface must be customized to containunique capture binding ligands. Alternatively, the use of captureextender ligands, particularly when the capture extender ligands arecapture extender probes (i.e. nucleic acids) may be used, that allow a“universal” surface, i.e. a surface containing a single type of captureprobe that can be used to detect any target sequence.

In a preferred embodiment, the capture binding ligands are added afterthe formation of the SAM ((4) above). This may be done in a variety ofways, as will be appreciated by those in the art. In one embodiment,conductive oligomers with terminal functional groups are made, withpreferred embodiments utilizing activated carboxylates andisothiocyanates, that will react with primary amines that are put ontothe binding ligand, using an activated carboxylate and nucleic acid,although other capture ligands may be attached in this way as well.These two reagents have the advantage of being stable in aqueoussolution, yet react with primary alkylamines. This allows the spottingof probes (either capture or detection probes, or both) using knownmethods (ink jet, spotting, etc.) onto the surface.

In addition, there are a number of non-nucleic acid methods that can beused to immobilize a capture binding ligand on a surface. For example,binding partner pairs can be utilized; i.e. one binding partner isattached to the terminus of the conductive oligomer, and the other tothe end of the binding ligand. This may also be done without using anucleic acid capture probe; that is, one binding partner serves as thecapture probe and the other is attached to either the target sequence ora capture extender probe. That is, either the target sequence comprisesthe binding partner, or a capture extender probe that will hybridize tothe target sequence comprises the binding partner. Suitable bindingpartner pairs include, but are not limited to, hapten pairs such asbiotin/streptavidin; antigens/antibodies; NTA/histidine tags; etc. Ingeneral, smaller binding partners are preferred.

In a preferred embodiment, when the target sequence itself is modifiedto contain a binding partner, the binding partner is attached via amodified nucleotide that can be enzymatically attached to the targetsequence, for example during a PCR target amplification step.Alternatively, the binding partner should be easily attached to thetarget sequence.

Alternatively, a capture extender probe may be utilized that has anucleic acid portion for hybridization to the target as well as abinding partner (for example, the capture extender probe may comprise anon-nucleic acid portion such as an alkyl linker that is used to attacha binding partner). In this embodiment, it may be desirable tocross-link the double-stranded nucleic acid of the target and captureextender probe for stability, for example using psoralen as is known inthe art.

In one embodiment, the target is not bound to the electrode surfaceusing capture binding ligands. In this embodiment, what is important, asfor all the assays herein, is that excess label probes be removed priorto detection and that the assay complex (comprising the recruitmentlinker) be in proximity to the surface. As will be appreciated by thosein the art, this may be accomplished in other ways. For example, theassay complex may be present on beads that are added to the electrodecomprising the monolayer. The recruitment linkers comprising the ETMsmay be placed in proximity to the conductive oligomer surface usingtechniques well known in the art, including gravity settling of thebeads on the surface, electrostatic or magnetic interactions betweenbead components and the surface, using binding partner attachment asoutlined above. Alternatively, after the removal of excess reagents suchas excess label probes, the assay complex may be driven down to thesurface, for example by pulsing the system with a voltage sufficient todrive the assay complex to the surface.

However, preferred embodiments utilize assay complexes attached viacapture binding ligands.

For nucleic acid systems, a preferred embodiments utilize the targetsequence itself containing the ETMs. As discussed above, this may bedone using target sequences that have ETMs incorporated at any number ofpositions, as outlined above. In this embodiment, as for the others ofthe system, the 3′-5′ orientation of the probes and targets is chosen toget the ETM-containing structures (i.e. recruitment linkers or targetsequences) as close to the surface of the monolayer as possible, and inthe correct orientation. This may be done using attachment viainsulators or conductive oligomers. In addition, as will be appreciatedby those in the art, multiple capture probes can be utilized, either ina configuration wherein the 5′-3′ orientation of the capture probes isdifferent, or where “loops” of target form when multiples of captureprobes are used.

For nucleic acid systems, a preferred embodiments utilize the labelprobes directly hybridizing to the target sequences, as is generallydepicted in FIGS. 7D-7I. In these embodiments, the target sequence ispreferably, but not required to be, immobilized on the surface usingcapture probes, including capture extender probes. Label probes are thenused to bring the ETMs into proximity of the surface of the monolayercomprising conductive oligomers. In a preferred embodiment, multiplelabel probes are used; that is, label probes are designed such that theportion that hybridizes to the target sequence (labeled 41 in thefigures) can be different for a number of different label probes, suchthat amplification of the signal occurs, since multiple label probes canbind for every target sequence. Thus, as depicted in the figures, n isan integer of at least one. Depending on the sensitivity desired, thelength of the target sequence, the number of ETMs per label probe, etc.,preferred ranges of n are from 1 to 50, with from about 1 to about 20being particularly preferred, and from about 2 to about 5 beingespecially preferred. In addition, if “generic” label probes aredesired, label extender probes can be used as generally described belowfor use with amplifier probes.

As above, generally in this embodiment the configuration of the systemand the label probes (recruitment linkers) are designed to recruit theETMs as close as possible to the monolayer surface.

In a preferred embodiment, the label probes are bound to the targetanalyte indirectly. That is, the present invention finds use in novelcombinations of signal amplification technologies and electron transferdetection on electrodes, which may be particularly useful in sandwichhybridization assays, for nucleic acid detection. In these embodiments,the amplifier probes of the invention are bound to the target sequencein a sample either directly or indirectly. Since the amplifier probespreferably contain a relatively large number of amplification sequencesthat are available for binding of label probes, the detectable signal issignificantly increased, and allows the detection limits of the targetto be significantly improved. These label and amplifier probes, and thedetection methods described herein, may be used in essentially any knownnucleic acid hybridization formats, such as those in which the target isbound directly to a solid phase or in sandwich hybridization assays inwhich the target is bound to one or more nucleic acids that are in turnbound to the solid phase.

In general, these embodiments may be described as follows. An amplifierprobe is hybridized to the target sequence, either directly (e.g. FIG.7I), or through the use of a label extender probe (e.g. FIGS. 7N and7O), which serves to allow “generic” amplifier probes to be made. Thetarget sequence is preferably, but not required to be, immobilized onthe electrode using capture probes. Preferably, the amplifier probecontains a multiplicity of amplification sequences, although in someembodiments, as described below, the amplifier probe may contain only asingle amplification sequence. The amplifier probe may take on a numberof different forms; either a branched conformation, a dendrimerconformation, or a linear “string” of amplification sequences. Theseamplification sequences are used to form hybridization complexes withlabel probes, and the ETMs can be detected using the electrode.

Accordingly, the present invention provides assay complexes comprisingat least one amplifier probe. By “amplifier probe” or “nucleic acidmultimer” or “amplification multimer” or grammatical equivalents hereinis meant a nucleic acid probe that is used to facilitate signalamplification. Amplifier probes comprise at least a firstsingle-stranded nucleic acid probe sequence, as defined below, and atleast one single-stranded nucleic acid amplification sequence, with amultiplicity of amplification sequences being preferred. In someembodiments, it is possible to use amplifier binding ligands, that arenon-nucleic acid based but that comprise a plurality of binding sitesfor the later association/binding of label ligands comprisingrecruitment linkers. However, amplifier probes are preferred in nucleicacid systems.

Amplifier probes comprise a first probe sequence that is used, eitherdirectly or indirectly, to hybridize to the target sequence. That is,the amplifier probe itself may have a first probe sequence that issubstantially complementary to the target sequence (e.g. FIG. 7I), or ithas a first probe sequence that is substantially complementary to aportion of an additional probe, in this case called a label extenderprobe, that has a first portion that is substantially complementary tothe target sequence (e.g. FIG. 7N). In a preferred embodiment, the firstprobe sequence of the amplifier probe is substantially complementary tothe target sequence, as is generally depicted in FIG. 7I.

In general, as for all the probes herein, the first probe sequence is ofa length sufficient to give specificity and stability. Thus generally,the probe sequences of the invention that are designed to hybridize toanother nucleic acid (i.e. probe sequences, amplification sequences,portions or domains of larger probes) are at least about 5 nucleosideslong, with at least about 10 being preferred and at least about 15 beingespecially preferred.

In a preferred embodiment, the amplifier probes, or any of the otherprobes of the invention, may form hairpin stem-loop structures in theabsence of their target. The length of the stem double-stranded sequencewill be selected such that the hairpin structure is not favored in thepresence of target. The use of these type of probes, in the systems ofthe invention or in any nucleic acid detection systems, can result in asignificant decrease in non-specific binding and thus an increase in thesignal to noise ratio.

Generally, these hairpin structures comprise four components. The firstcomponent is a target binding sequence, i.e. a region complementary tothe target (which may be the sample target sequence or another probesequence to which binding is desired), that is about 10 nucleosideslong, with about 15 being preferred. The second component is a loopsequence, that can facilitate the formation of nucleic acid loops.Particularly preferred in this regard are repeats of GTC, which has beenidentified in Fragile X Syndrome as forming turns. (When PNA analogs areused, turns comprising proline residues may be preferred). Generally,from three to five repeats are used, with four to five being preferred.The third component is a self-complementary region, which has a firstportion that is complementary to a portion of the target sequence regionand a second portion that comprises a first portion of the label probebinding sequence. The fourth component is substantially complementary toa label probe (or other probe, as the case may be). The fourth componentfurther comprises a “sticky end”, that is, a portion that does nothybridize to any other portion of the probe, and preferably containsmost, if not all, of the ETMs. As will be appreciated by those in theart, the any or all of the probes described herein may be configured toform hairpins in the absence of their targets, including the amplifier,capture, capture extender, label and label extender probes.

In a preferred embodiment, several different amplifier probes are used,each with first probe sequences that will hybridize to a differentportion of the target sequence. That is, there is more than one level ofamplification; the amplifier probe provides an amplification of signaldue to a multiplicity of labelling events, and several differentamplifier probes, each with this multiplicity of labels, for each targetsequence is used. Thus, preferred embodiments utilize at least twodifferent pools of amplifier probes, each pool having a different probesequence for hybridization to different portions of the target sequence;the only real limitation on the number of different amplifier probeswill be the length of the original target sequence. In addition, it isalso possible that the different amplifier probes contain differentamplification sequences, although this is generally not preferred.

In a preferred embodiment, the amplifier probe does not hybridize to thesample target sequence directly, but instead hybridizes to a firstportion of a label extender probe, as is generally depicted in FIG. 7L.This is particularly useful to allow the use of “generic” amplifierprobes, that is, amplifier probes that can be used with a variety ofdifferent targets. This may be desirable since several of the amplifierprobes require special synthesis techniques. Thus, the addition of arelatively short probe as a label extender probe is preferred. Thus, thefirst probe sequence of the amplifier probe is substantiallycomplementary to a first portion or domain of a first label extendersingle-stranded nucleic acid probe. The label extender probe alsocontains a second portion or domain that is substantially complementaryto a portion of the target sequence. Both of these portions arepreferably at least about 10 to about 50 nucleotides in length, with arange of about 15 to about 30 being preferred. The terms “first” and“second” are not meant to confer an orientation of the sequences withrespect to the 5′-3′ orientation of the target or probe sequences. Forexample, assuming a 5′-3′ orientation of the complementary targetsequence, the first portion may be located either 5′ to the secondportion, or 3′ to the second portion. For convenience herein, the orderof probe sequences are generally shown from left to right.

In a preferred embodiment, more than one label extender probe-amplifierprobe pair may be used, that is, n is more than 1. That is, a pluralityof label extender probes may be used, each with a portion that issubstantially complementary to a different portion of the targetsequence; this can serve as another level of amplification. Thus, apreferred embodiment utilizes pools of at least two label extenderprobes, with the upper limit being set by the length of the targetsequence.

In a preferred embodiment, more than one label extender probe is usedwith a single amplifier probe to reduce non-specific binding, as isdepicted in FIG. 7O and generally outlined in U.S. Pat. No. 5,681,697,incorporated by reference herein. In this embodiment, a first portion ofthe first label extender probe hybridizes to a first portion of thetarget sequence, and the second portion of the first label extenderprobe hybridizes to a first probe sequence of the amplifier probe. Afirst portion of the second label extender probe hybridizes to a secondportion of the target sequence, and the second portion of the secondlabel extender probe hybridizes to a second probe sequence of theamplifier probe. These form structures sometimes referred to as“cruciform” structures or configurations, and are generally done toconfer stability when large branched or dendrimeric amplifier probes areused.

In addition, as will be appreciated by those in the art, the labelextender probes may interact with a preamplifier probe, described below,rather than the amplifier probe directly.

Similarly, as outlined above, a preferred embodiment utilizes severaldifferent amplifier probes, each with first probe sequences that willhybridize to a different portion of the label extender probe. Inaddition, as outlined above, it is also possible that the differentamplifier probes contain different amplification sequences, althoughthis is generally not preferred.

In addition to the first probe sequence, the amplifier probe alsocomprises at least one amplification sequence. An “amplificationsequence” or “amplification segment” or grammatical equivalents hereinis meant a sequence that is used, either directly or indirectly, to bindto a first portion of a label probe as is more fully described below.Preferably, the amplifier probe comprises a multiplicity ofamplification sequences, with from about 3 to about 1000 beingpreferred, from about 10 to about 100 being particularly preferred, andabout 50 being especially preferred. In some cases, for example whenlinear amplifier probes are used, from 1 to about 20 is preferred withfrom about 5 to about 10 being particularly preferred. Again, whennon-nucleic acid amplifier moieties are used, the amplification segmentcan bind label ligands.

The amplification sequences may be linked to each other in a variety ofways, as will be appreciated by those in the art. They may be covalentlylinked directly to each other, or to intervening sequences or chemicalmoieties, through nucleic acid linkages such as phosphodiester bonds,PNA bonds, etc., or through interposed linking agents such amino acid,carbohydrate or polyol bridges, or through other cross-linking agents orbinding partners. The site(s) of linkage may be at the ends of asegment, and/or at one or more internal nucleotides in the strand. In apreferred embodiment, the amplification sequences are attached vianucleic acid linkages.

In a preferred embodiment, branched amplifier probes are used, as aregenerally described in U.S. Pat. No. 5,124,246, hereby incorporated byreference. Branched amplifier probes may take on “fork-like” or“comb-like” conformations. “Fork-like” branched amplifier probesgenerally have three or more oligonucleotide segments emanating from apoint of origin to form a branched structure. The point of origin may beanother nucleotide segment or a multifunctional molecule to whcih atleast three segments can be covalently or tightly bound. “Comb-like”branched amplifier probes have a linear backbone with a multiplicity ofsidechain oligonucleotides extending from the backbone. In eitherconformation, the pendant segments will normally depend from a modifiednucleotide or other organic moiety having the appropriate functionalgroups for attachment of oligonucleotides. Furthermore, in eitherconformation, a large number of amplification sequences are availablefor binding, either directly or indirectly, to detection probes. Ingeneral, these structures are made as is known in the art, usingmodified multifunctional nucleotides, as is described in U.S. Pat. Nos.5,635,352 and 5,124,246, among others.

In a preferred embodiment, dendrimer amplifier probes are used, as aregenerally described in U.S. Pat. No. 5,175,270, hereby expresslyincorporated by reference. Dendrimeric amplifier probes haveamplification sequences that are attached via hybridization, and thushave portions of double-stranded nucleic acid as a component of theirstructure. The outer surface of the dendrimer amplifier probe has amultiplicity of amplification sequences.

In a preferred embodiment, linear amplifier probes are used, that haveindividual amplification sequences linked end-to-end either directly orwith short intervening sequences to form a polymer. As with the otheramplifier configurations, there may be additional sequences or moietiesbetween the amplification sequences. In addition, as outlined herein,linear amplification probes may form hairpin stem-loop structures.

In one embodiment, the linear amplifier probe has a single amplificationsequence. This may be useful when cycles of hybridization/disassociationoccurs, forming a pool of amplifier probe that was hybridized to thetarget and then removed to allow more probes to bind, or when largenumbers of ETMs are used for each label probe. However, in a preferredembodiment, linear amplifier probes comprise a multiplicity ofamplification sequences.

In addition, the amplifier probe may be totally linear, totallybranched, totally dendrimeric, or any combination thereof.

The amplification sequences of the amplifier probe are used, eitherdirectly or indirectly, to bind to a label probe to allow detection. Ina preferred embodiment, the amplification sequences of the amplifierprobe are substantially complementary to a first portion of a labelprobe. Alternatively, amplifier extender probes are used, that have afirst portion that binds to the amplification sequence and a secondportion that binds to the first portion of the label probe.

In addition, the compositions of the invention may include“preamplifier” molecules, which serves a bridging moiety between thelabel extender molecules and the amplifier probes. In this way, moreamplifier and thus more ETMs are ultimately bound to the detectionprobes. Preamplifier molecules may be either linear or branched, andtypically contain in the range of about 30-3000 nucleotides. Thereactions outlined below may be accomplished in a variety of ways, aswill be appreciated by those in the art. Components of the reaction maybe added simultaneously, or sequentially, in any order, with preferredembodiments outlined below. In addition, the reaction may include avariety of other reagents may be included in the assays. These includereagents like salts, buffers, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal hybridizationand detection, and/or reduce non-specific or background interactions.Also reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents,etc., may be used, depending on the sample preparation methods andpurity of the target.

Generally, the methods are as follows. In a preferred embodiment, thetarget is initially immobilized or attached to the electrode. Fornucleic acids, this is done by forming a hybridization complex between acapture probe and a portion of the target sequence. A preferredembodiment utilizes capture extender probes; in this embodiment, ahybridization complex is formed between a portion of the target sequenceand a first portion of a capture extender probe, and an additionalhybridization complex between a second portion of the capture extenderprobe and a portion of the capture probe. Additional preferredembodiments utilize additional capture probes, thus forming ahybridization complex between a portion of the target sequence and afirst portion of a second capture extender probe, and an additionalhybridization complex between a second portion of the second captureextender probe and a second portion of the capture probe. Non-nucleicacid embodiments utilize capture binding ligands and optional captureextender ligands.

Alternatively, the attachment of the target sequence to the electrode isdone simultaneously with the other reactions.

The method proceeds with the introduction of amplifier probes, ifutilized. In a preferred embodiment, the amplifier probe comprises afirst probe sequence that is substantially complementary to a portion ofthe target sequence, and at least one amplification sequence.

In one embodiment, the first probe sequence of the amplifier probe ishybridized to the target sequence, and any unhybridized amplifier probeis removed. This will generally be done as is known in the art, anddepends on the type of assay. When the target sequence is immobilized ona surface such as an electrode, the removal of excess reagents generallyis done via one or more washing steps, as will be appreciated by thosein the art. In this embodiment, the target may be immobilized on anysolid support. When the target sequence is not immobilized on a surface,the removal of excess reagents such as the probes of the invention maybe done by adding beads (i.e. solid support particles) that containcomplementary sequences to the probes, such that the excess probes bindto the beads. The beads can then be removed, for example bycentrifugation, filtration, the application of magnetic or electrostaticfields, etc.

The reaction mixture is then subjected to conditions (temperature, highsalt, changes in pH, etc.) under which the amplifier probe disassociatesfrom the target sequence, and the amplifier probe is collected. Theamplifier probe may then be added to an electrode comprising captureprobes for the amplifier probes, label probes added, and detection isachieved.

In a preferred embodiment, a larger pool of probe is generated by addingmore amplifier probe to the target sequence and thehybridization/disassociation reactions are repeated, to generate alarger pool of amplifier probe. This pool of amplifier probe is thenadded to an electrode comprising amplifier capture probes, label probesadded, and detection proceeds.

In this embodiment, it is preferred that the target analyte beimmobilized on a solid support, including an electrode, using themethods described herein; although as will be appreciated by those inthe art, alternate solid support attachment technologies may be used,such as attachment to glass, polymers, etc. It is possible to do thereaction on one solid support and then add the pooled amplifier probe toan electrode for detection.

In a preferred embodiment, the amplifier probe comprises a multiplicityof amplification sequences.

In one embodiment, the first probe sequence of the amplifier probe ishybridized to the target sequence, and any unhybridized amplifier probeis removed. Again, preferred embodiments utilize immobilized targetsequences, wherein the target sequences are immobilized by hybridizationwith capture probes that are attached to the electrode, or hybridizationto capture extender probes that in turn hybridize with immobilizedcapture probes as is described herein. Generally, in these embodiments,the capture probes and the detection probes are immobilized on theelectrode, generally at the same “address”.

In a preferred embodiment, the first probe sequence of the amplifierprobe is hybridized to a first portion of at least one label extenderprobe, and a second portion of the label extender probe is hybridized toa portion of the target sequence. Other preferred embodiments utilizemore than one label extender probe, as is generally shown in FIG. 7O.

In a preferred embodiment, the amplification sequences of the amplifierprobe are used directly for detection, by hybridizing at least one labelprobe sequence.

The invention thus provides assay complexes that minimally comprise atarget sequence and a label probe. “Assay complex” herein is meant thecollection of binding complexes comprising capture binding ligands,target analytes (or analogs, as described below) and label moietiescomprising recruitment linkers that allows detection. The composition ofthe assay complex depends on the use of the different componentsoutlined herein. Thus, the assay complex comprises the capture probe andthe target sequence. The assay complexes may also include captureextender ligands (including probes), label extender ligands, andamplifier ligands, as outlined herein, depending on the configurationused.

The assays are generally run under conditions which allows formation ofthe assay complex only in the presence of target. Stringency can becontrolled by altering a step parameter that is a thermodynamicvariable, including, but not limited to, temperature, formamideconcentration, salt concentration, chaotropic salt concentration pH,organic solvent concentration, etc.

These parameters may also be used to control non-specific binding fornucleic acids, as is generally outlined in U.S. Pat. No. 5,681,697. Thusit may be desirable to perform certain steps at higher stringencyconditions; for example, when an initial hybridization step is donebetween the target sequence and the label extender and capture extenderprobes. Running this step at conditions which favor specific binding canallow the reduction of non-specific binding.

In a preferred embodiment, when all of the components outlined hereinare used, a preferred method for nucleic acid detection is as follows.Single-stranded target sequence is incubated under hybridizationconditions with the capture extender probes and the label extenderprobes. A preferred embodiment does this reaction in the presence of theelectrode with immobilized capture probes, although this may also bedone in two steps, with the initial incubation and the subsequentaddition to the electrode. Excess reagents are washed off, and amplifierprobes are then added. If preamplifier probes are used, they may beadded either prior to the amplifier probes or simultaneously with theamplifier probes. Excess reagents are washed off, and label probes arethen added. Excess reagents are washed off, and detection proceeds asoutlined below.

In one embodiment, a number of capture probes (or capture probes andcapture extender probes) that are each substantially complementary to adifferent portion of the target sequence are used.

Again, as outlined herein, when amplifier probes are used, the system isgenerally configured such that upon label probe binding, the recruitmentlinkers comprising the ETMs are placed in proximity to the monolayersurface. Thus for example, when the ETMs are attached via “dendrimer”type structures as outlined herein, the length of the linkers from thenucleic acid point of attachment to the ETMs may vary, particularly withthe length of the capture probe when capture extender probes are used.That is, longer capture probes, with capture extenders, can result inthe target sequences being “held” further away from the surface than forshorter capture probes. Adding extra linking sequences between the probenucleic acid and the ETMs can result in the ETMs being spatially closerto the surface, giving better results.

In addition, if desirable, nucleic acids utilized in the invention mayalso be ligated together prior to detection, if applicable, by usingstandard molecular biology techniques such as the use of a ligase.Similarly, if desirable for stability, cross-linking agents may be addedto hold the structures stable.

Other embodiments of the invention utilize different steps. For example,competitive assays may be run. In this embodiment, the target analyte ina sample may be replaced by a target analyte analog comprising a portionthat either comprises a recruitment linker or can indirectly bind arecruitment linker. This may be done as is known in the art, for exampleby using affinity chromatography techniques that exchange the analog forthe analyte, leaving the analyte bound and the analog free to interactwith the capture binding ligands on the electrode surface. This isgenerally depicted in FIG. 6A.

Alternatively, a preferred embodiment utilizes a competitive bindingassay when the solution binding ligand comprises a directly orindirectly associated recruitment linker comprising ETMs. In thisembodiment, a target analyte or target analyte analog that will bind thesolution binding ligand is attached to the surface. The solution bindingligand will bind to the surface bound analyte and give a signal. Uponintroduction of the target analyte of the sample, a proportion of thesolution binding ligand will dissociate from the surface bound targetand bind to the incoming target analyte. Thus, a loss of signalproportional to the amount of target analyte in the sample is seen.

The compositions of the invention are generally synthesized as outlinedbelow, generally utilizing techniques well known in the art. As will beappreciated by those in the art, many of the techniques outlined beloware directed to nucleic acids containing a ribose-phosphate backbone.However, as outlined above, many alternate nucleic acid analogs may beutilized, some of which may not contain either ribose or phosphate inthe backbone. In these embodiments, for attachment at positions otherthan the base, attachment is done as will be appreciated by those in theart, depending on the backbone. Thus, for example, attachment can bemade at the carbon atoms of the PNA backbone, as is described below, orat either terminus of the PNA.

The compositions may be made in several ways. A preferred method firstsynthesizes a conductive oligomer attached to a nucleoside, withaddition of additional nucleosides to form the capture probe followed byattachment to the electrode. Alternatively, the whole capture probe maybe made and then the completed conductive oligomer added, followed byattachment to the electrode. Alternatively, a monolayer of conductiveoligomer (some of which have functional groups for attachment of captureprobes) is attached to the electrode first, followed by attachment ofthe capture probe. The latter two methods may be preferred whenconductive oligomers are used which are not stable in the solvents andunder the conditions used in traditional nucleic acid synthesis.

In a preferred embodiment, the compositions of the invention are made byfirst forming the conductive oligomer covalently attached to thenucleoside, followed by the addition of additional nucleosides to form acapture probe nucleic acid, with the last step comprising the additionof the conductive oligomer to the electrode.

The attachment of the conductive oligomer to the nucleoside may be donein several ways. In a preferred embodiment, all or part of theconductive oligomer is synthesized first (generally with a functionalgroup on the end for attachment to the electrode), which is thenattached to the nucleoside. Additional nucleosides are then added asrequired, with the last step generally being attachment to theelectrode. Alternatively, oligomer units are added one at a time to thenucleoside, with addition of additional nucleosides and attachment tothe electrode. A number of representative syntheses are shown in theFigures of WO 98/20162, PCT US98/12430, PCT US99/01705 and PCTUS99/01703, all of which are expressly incorporated by reference.

The conductive oligomer is then attached to a nucleoside that maycontain one (or more) of the oligomer units, attached as depictedherein.

In a preferred embodiment, attachment is to a ribose of theribose-phosphate backbone in a number of ways, including attachment viaamide and amine linkages. In a preferred embodiment, there is at least amethylene group or other short aliphatic alkyl groups (as a Z group)between the nitrogen attached to the ribose and the aromatic ring of theconductive oligomer.

Alternatively, attachment is via a phosphate of the ribose-phosphatebackbone.

In a preferred embodiment, attachment is via the base, and can includeacetylene linkages and amide linkages. In a preferred embodiment,protecting groups may be added to the base prior to addition of theconductive oligomers. In addition, the palladium cross-couplingreactions may be altered to prevent dimerization problems; i.e. twoconductive oligomers dimerizing, rather than coupling to the base.

Alternatively, attachment to the base may be done by making thenucleoside with one unit of the oligomer, followed by the addition ofothers.

Once the modified nucleosides are prepared, protected and activated,prior to attachment to the electrode, they may be incorporated into agrowing oligonucleotide by standard synthetic techniques (Gait,Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, UK1984; Eckstein) in several ways.

In one embodiment, one or more modified nucleosides are converted to thetriphosphate form and incorporated into a growing oligonucleotide chainby using standard molecular biology techniques such as with the use ofthe enzyme DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, TaqDNA polymerase, reverse transcriptase, and RNA polymerases. For theincorporation of a 3′ modified nucleoside to a nucleic acid, terminaldeoxynucleotidyltransferase may be used. (Ratliff, Terminaldeoxynucleotidyltransferase. In The Enzymes, Vol 14A. P.D. Boyer ed. pp105-118. Academic Press, San Diego, Calif. 1981). Thus, the presentinvention provides deoxyribonucleoside triphosphates comprising acovalently attached ETM. Preferred embodiments utilize ETM attachment tothe base or the backbone, such as the ribose (preferably in the 2′position), as is generally depicted below in Structures 42 and 43:

Thus, in some embodiments, it may be possible to generate the nucleicacids comprising ETMs in situ. For example, a target sequence canhybridize to a capture probe (for example on the surface) in such a waythat the terminus of the target sequence is exposed, i.e. unhybridized.The addition of enzyme and triphosphate nucleotides labelled with ETMsallows the in situ creation of the label. Similarly, using labelednucleotides recognized by polymerases can allow simultaneous PCR anddetection; that is, the target sequences are generated in situ.

In a preferred embodiment, the modified nucleoside is converted to thephosphoramidite or H-phosphonate form, which are then used insolid-phase or solution syntheses of oligonucleotides. In this way themodified nucleoside, either for attachment at the ribose (i.e. amino- orthiol-modified nucleosides) or the base, is incorporated into theoligonucleotide at either an internal position or the 5′ terminus. Thisis generally done in one of two ways. First, the 5′ position of theribose is protected with 4′,4-dimethoxytrityl (DMT) followed by reactionwith either 2-cyanoethoxy-bis-diisopropylaminophosphine in the presenceof diisopropylammonium tetrazolide, or by reaction withchlorodiisopropylamino 2′-cyanoethyoxyphosphine, to give thephosphoramidite as is known in the art; although other techniques may beused as will be appreciated by those in the art. See Gait, supra;Caruthers, Science 230:281 (1985), both of which are expresslyincorporated herein by reference.

For attachment of a group to the 3′ terminus, a preferred methodutilizes the attachment of the modified nucleoside (or the nucleosidereplacement) to controlled pore glass (CPG) or other oligomericsupports. In this embodiment, the modified nucleoside is protected atthe 5′ end with DMT, and then reacted with succinic anhydride withactivation. The resulting succinyl compound is attached to CPG or otheroligomeric supports as is known in the art. Further phosphoramiditenucleosides are added, either modified or not, to the 5′ end afterdeprotection. Thus, the present invention provides conductive oligomersor insulators covalently attached to nucleosides attached to solidoligomeric supports such as CPG, and phosphoramidite derivatives of thenucleosides of the invention.

The invention further provides methods of making label probes withrecruitment linkers comprising ETMs. These synthetic reactions willdepend on the character of the recruitment linker and the method ofattachment of the ETM, as will be appreciated by those in the art. Fornucleic acid recruitment linkers, the label probes are generally made asoutlined herein with the incorporation of ETMs at one or more positions.When a transition metal complex is used as the ETM, synthesis may occurin several ways. In a preferred embodiment, the ligand(s) are added to anucleoside, followed by the transition metal ion, and then thenucleoside with the transition metal complex attached is added to anoligonucleotide, i.e. by addition to the nucleic acid synthesizer.Alternatively, the ligand(s) may be attached, followed by incorportationinto a growing oligonucleotide chain, followed by the addition of themetal ion.

In a preferred embodiment, ETMs are attached to a ribose of theribose-phosphate backbone. This is generally done as is outlined hereinfor conductive oligomers, as described herein, and in PCT publication WO95/15971, using amino-modified or oxo-modified nucleosides, at eitherthe 2′ or 3′ position of the ribose. The amino group may then be usedeither as a ligand, for example as a transition metal ligand forattachment of the metal ion, or as a chemically functional group thatcan be used for attachment of other ligands or organic ETMs, for examplevia amide linkages, as will be appreciated by those in the art. Forexample, the examples describe the synthesis of nucleosides with avariety of ETMs attached via the ribose.

In a preferred embodiment, ETMs are attached to a phosphate of theribose-phosphate backbone. As outlined herein, this may be done usingphosphodiester analogs such as phosphoramidite bonds, see generally PCTpublication WO 95/15971, or the figures.

Attachment to alternate backbones, for example peptide nucleic acids oralternate phosphate linkages will be done as will be appreciated bythose in the art.

In a preferred embodiment, ETMs are attached to a base of thenucleoside. This may be done in a variety of ways. In one embodiment,amino groups of the base, either naturally occurring or added as isdescribed herein (see the figures, for example), are used either asligands for transition metal complexes or as a chemically functionalgroup that can be used to add other ligands, for example via an amidelinkage, or organic ETMs. This is done as will be appreciated by thosein the art. Alternatively, nucleosides containing halogen atoms attachedto the heterocyclic ring are commercially available. Acetylene linkedligands may be added using the halogenated bases, as is generally known;see for example, Tzalis et al., Tetrahedron Lett. 36(34):6017-6020(1995); Tzalis et al., Tetrahedron Left. 36(2):3489-3490 (1995); andTzalis et al., Chem. Communications (in press) 1996, all of which arehereby expressly incorporated by reference. See also PCT/US99/10104which describes the synthesis of metallocenes (in this case, ferrocene)attached via acetylene linkages to the bases.

In one embodiment, the nucleosides are made with transition metalligands, incorporated into a nucleic acid, and then the transition metalion and any remaining necessary ligands are added as is known in theart. In an alternative embodiment, the transition metal ion andadditional ligands are added prior to incorporation into the nucleicacid.

Once the nucleic acids of the invention are made, with a covalentlyattached attachment linker (i.e. either an insulator or a conductiveoligomer), the attachment linker is attached to the electrode. Themethod will vary depending on the type of electrode used. As isdescribed herein, the attachment linkers are generally made with aterminal “A” linker to facilitate attachment to the electrode. For thepurposes of this application, a sulfur-gold attachment is considered acovalent attachment.

In a preferred embodiment, conductive oligomers, insulators, andattachment linkers are covalently attached via sulfur linkages to theelectrode. However, surprisingly, traditional protecting groups for useof attaching molecules to gold electrodes are generally not ideal foruse in both synthesis of the compositions described herein and inclusionin oligonucleotide synthetic reactions. Accordingly, the presentinvention provides novel methods for the attachment of conductiveoligomers to gold electrodes, utilizing unusual protecting groups,including ethylpyridine, and trimethylsilylethyl. However, as will beappreciated by those in the art, when the conductive oligomers do notcontain nucleic acids, traditional protecting groups such as acetylgroups and others may be used. See Greene et al., supra.

This may be done in several ways. In a preferred embodiment, the subunitof the conductive oligomer which contains the sulfur atom for attachmentto the electrode is protected with an ethyl-pyridine ortrimethylsilylethyl group. For the former, this is generally done bycontacting the subunit containing the sulfur atom (preferably in theform of a sulfhydryl) with a vinyl pyridine group or vinyltrimethylsilylethyl group under conditions whereby an ethylpyridinegroup or trimethylsilylethyl group is added to the sulfur atom.

This subunit also generally contains a functional moiety for attachmentof additional subunits, and thus additional subunits are attached toform the conductive oligomer. The conductive oligomer is then attachedto a nucleoside, and additional nucleosides attached. The protectinggroup is then removed and the sulfur-gold covalent attachment is made.Alternatively, all or part of the conductive oligomer is made, and theneither a subunit containing a protected sulfur atom is added, or asulfur atom is added and then protected. The conductive oligomer is thenattached to a nucleoside, and additional nucleosides attached.Alternatively, the conductive oligomer attached to a nucleic acid ismade, and then either a subunit containing a protected sulfur atom isadded, or a sulfur atom is added and then protected. Alternatively, theethyl pyridine protecting group may be used as above, but removed afterone or more steps and replaced with a standard protecting group like adisulfide. Thus, the ethyl pyridine or trimethylsilylethyl group mayserve as the protecting group for some of the synthetic reactions, andthen removed and replaced with a traditional protecting group.

By “subunit” of a conductive polymer herein is meant at least the moietyof the conductive oligomer to which the sulfur atom is attached,although additional atoms may be present, including either functionalgroups which allow the addition of additional components of theconductive oligomer, or additional components of the conductiveoligomer. Thus, for example, when Structure 1 oligomers are used, asubunit comprises at least the first Y group.

A preferred method comprises 1) adding an ethyl pyridine ortrimethylsilylethyl protecting group to a sulfur atom attached to afirst subunit of a conductive oligomer, generally done by adding a vinylpyridine or trimethylsilylethyl group to a sulfhydryl; 2) addingadditional subunits to form the conductive oligomer; 3) adding at leasta first nucleoside to the conductive oligomer; 4) adding additionalnucleosides to the first nucleoside to form a nucleic acid; 5) attachingthe conductive oligomer to the gold electrode. This may also be done inthe absence of nucleosides.

The above methods may also be used to attach insulator molecules to agold electrode, and moieties comprising capture binding ligands.

In a preferred embodiment, a monolayer comprising conductive oligomers(and preferably insulators) is added to the electrode. Generally, thechemistry of addition is similar to or the same as the addition ofconductive oligomers to the electrode, i.e. using a sulfur atom forattachment to a gold electrode, etc. Compositions comprising monolayersin addition to the conductive oligomers covalently attached to nucleicacids may be made in at least one of five ways: (1) addition of themonolayer, followed by subsequent addition of the attachmentlinker-nucleic acid complex; (2) addition of theattachmentlinker-nucleic acid complex followed by addition of the monolayer; (3)simultaneous addition of the monolayer and attachment linker-nucleicacid complex; (4) formation of a monolayer (using any of 1, 2 or 3)which includes attachment linkers which terminate in a functional moietysuitable for attachment of a completed nucleic acid; or (5) formation ofa monolayer which includes attachment linkers which terminate in afunctional moiety suitable for nucleic acid synthesis, i.e. the nucleicacid is synthesized on the surface of the monolayer as is known in theart. Such suitable functional moieties include, but are not limited to,nucleosides, amino groups, carboxyl groups, protected sulfur moieties,or hydroxyl groups for phosphoramidite additions. The examples describethe formation of a monolayer on a gold electrode using the preferredmethod (1).

In a preferred embodiment, the nucleic acid is a peptide nucleic acid oranalog. In this embodiment, the invention provides peptide nucleic acidswith at least one covalently attached ETM or attachment linker. In apreferred embodiment, these moieties are covalently attached to anmonomeric subunit of the PNA. By “monomeric subunit of PNA” herein ismeant the —NH—CH₂CH₂—N(COCH₂-Base)—CH₂—CO— monomer, or derivatives(herein included within the definition of “nucleoside”) of PNA. Forexample, the number of carbon atoms in the PNA backbone may be altered;see generally Nielsen et al., Chem. Soc. Rev. 1997 page 73, whichdiscloses a number of PNA derivatives, herein expressly incorporated byreference. Similarly, the amide bond linking the base to the backbonemay be altered; phosphoramide and sulfuramide bonds may be used.Alternatively, the moieties are attached to an internal monomericsubunit. By “internal” herein is meant that the monomeric subunit is noteither the N-terminal monomeric subunit or the C-terminal monomericsubunit. In this embodiment, the moieties can be attached either to abase or to the backbone of the monomeric subunit. Attachment to the baseis done as outlined herein or known in the literature. In general, themoieties are added to a base which is then incorporated into a PNA asoutlined herein. The base may be either protected, as required forincorporation into the PNA synthetic reaction, or derivatized, to allowincorporation, either prior to the addition of the chemical substituentor afterwards. The bases can then be incorporated into monomericsubunits.

In a preferred embodiment, the moieties are covalently attached to thebackbone of the PNA monomer. The attachment is generally to one of theunsubstituted carbon atoms of the monomeric subunit, preferably theα-carbon of the backbone, although attachment at either of the carbon 1or 2 positions, or the α-carbon of the amide bond linking the base tothe backbone may be done. In the case of PNA analogs, other carbons oratoms may be substituted as well. In a preferred embodiment, moietiesare added at the a-carbon atoms, either to a terminal monomeric subunitor an internal one.

In this embodiment, a modified monomeric subunit is synthesized with anETM or an attachment linker, or a functional group for its attachment,and then the base is added and the modified monomer can be incorporatedinto a growing PNA chain.

Once generated, the monomeric subunits with covalently attached moietiesare incorporated into a PNA using the techniques outlined in Will etal., Tetrahedron 51(44):12069-12082 (1995), and Vanderlaan et al., Tett.Let. 38:2249-2252 (1997), both of which are hereby expresslyincorporated in their entirety. These procedures allow the addition ofchemical substituents to peptide nucleic acids without destroying thechemical substituents.

As will be appreciated by those in the art, electrodes may be made thathave any combination of nucleic acids, conductive oligomers andinsulators.

The compositions of the invention may additionally contain one or morelabels at any position. By “label” herein is meant an element (e.g. anisotope) or chemical compound that is attached to enable the detectionof the compound. Preferred labels are radioactive isotopic labels, andcolored or fluorescent dyes. The labels may be incorporated into thecompound at any position. In addition, the compositions of the inventionmay also contain other moieties such as cross-linking agents tofacilitate cross-linking of the target-probe complex. See for example,Lukhtanov et al., Nucl. Acids. Res. 24(4):683 (1996) and Tabone et al.,Biochem. 33:375 (1994), both of which are expressly incorporated byreference.

Once made, the compositions find use in a number of applications, asdescribed herein. In particular, the compositions of the invention finduse in target analyte assays. As will be appreciated by those in theart, electrodes can be made that have a single species of bindingligands such as nucleic acid, i.e. a single binding ligand, or multiplebinding ligand species.

In addition, as outlined herein, the use of a solid support such as anelectrode enables the use of these probes in an array form. The use ofoligonucleotide arrays are well known in the art, and the methods andcompositions herein allow the use of array formats for other targetanalytes as well. In addition, techniques are known for “addressing”locations within an electrode and for the surface modification ofelectrodes. Thus, in a preferred embodiment, arrays of different bindingligands are laid down on the electrode, each of which are covalentlyattached to the electrode via a conductive linker. In this embodiment,the number of different species may vary widely, from one to thousands,with from about 4 to about 100,000 being preferred, and from about 10 toabout 10,000 being particularly preferred.

The invention finds use in the screening of candidate bioactive agentsfor therapeutic agents that can alter the binding of the analyte to thebinding ligand, and thus may be involved in biological function. Theterm “agent” or “exogeneous compound” as used herein describes anymolecule, e.g., protein, oligopeptide, small organic molecule,polysaccharide, polynucleotide, etc., with the capability of directly orindirectly altering target analyte binding. Generally a plurality ofassay mixtures are run in parallel with different agent concentrationsto obtain a differential response to the various concentrations.Typically, one of these concentrations serves as a negative control,i.e., at zero concentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof. Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Altematively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

Candidate agents may be added either before or after the target analyte.

Once the assay complexes of the invention are made, that minimallycomprise a target sequence and a label probe, detection proceeds withelectronic initiation. Without being limited by the mechanism or theory,detection is based on the transfer of electrons from the ETM to theelectrode.

Detection of electron transfer, i.e. the presence of the ETMs, isgenerally initiated electronically, with voltage being preferred. Apotential is applied to the assay complex. Precise control andvariations in the applied potential can be via a potentiostat and eithera three electrode system (one reference, one sample (or working) and onecounter electrode) or a two electrode system (one sample and one counterelectrode). This allows matching of applied potential to peak potentialof the system which depends in part on the choice of ETMs and in part onthe conductive oligomer used, the composition and integrity of themonolayer, and what type of reference electrode is used. As describedherein, ferrocene is a preferred ETM.

In a preferred embodiment, a co-reductant or co-oxidant (collectively,co-redoxant) is used, as an additional electron source or sink. Seegenerally Sato et al., Bull. Chem. Soc. Jpn 66:1032 (1993); Uosaki etal., Electrochimica Acta 36:1799 (1991); and Alleman et al., J. Phys.Chem 100:17050 (1996); all of which are incorporated by reference.

In a preferred embodiment, an input electron source in solution is usedin the initiation of electron transfer, preferably when initiation anddetection are being done using DC current or at AC frequencies wherediffusion is not limiting. In general, as will be appreciated by thosein the art, preferred embodiments utilize monolayers that contain aminimum of “holes”, such that short-circuiting of the system is avoided.This may be done in several general ways. In a preferred embodiment, aninput electron source is used that has a lower or similar redoxpotential than the ETM of the label probe. Thus, at voltages above theredox potential of the input electron source, both the ETM and the inputelectron source are oxidized and can thus donate electrons; the ETMdonates an electron to the electrode and the input source donates to theETM. For example, ferrocene, as a ETM attached to the compositions ofthe invention as described in the examples, has a redox potential ofroughly 200 mV in aqueous solution (which can change significantlydepending on what the ferrocene is bound to, the manner of the linkageand the presence of any substitution groups). Ferrocyanide, an electronsource, has a redox potential of roughly 200 mV as well (in aqueoussolution). Accordingly, at or above voltages of roughly 200 mV,ferrocene is converted to ferricenium, which then transfers an electronto the electrode. Now the ferricyanide can be oxidized to transfer anelectron to the ETM. In this way, the electron source (or co-reductant)serves to amplify the signal generated in the system, as the electronsource molecules rapidly and repeatedly donate electrons to the ETMattached to the nucleic acid. The rate of electron donation oracceptance will be limited by the rate of diffusion of the co-reductant,the electron transfer between the co-reductant and the ETM, which inturn is affected by the concentration and size, etc.

Alternatively, input electron sources that have lower redox potentialsthan the ETM are used. At voltages less than the redox potential of theETM, but higher than the redox potential of the electron source, theinput source such as ferrocyanide is unable to be oxided and thus isunable to donate an electron to the ETM; i.e. no electron transferoccurs. Once ferrocene is oxidized, then there is a pathway for electrontransfer.

In an alternate preferred embodiment, an input electron source is usedthat has a higher redox potential than the ETM of the label probe. Forexample, luminol, an electron source, has a redox potential of roughly720 mV. At voltages higher than the redox potential of the ETM, butlower than the redox potential of the electron source, i.e. 200-720 mV,the ferrocene is oxided, and transfers a single electron to theelectrode via the conductive oligomer. However, the ETM is unable toaccept any electrons from the luminol electron source, since thevoltages are less than the redox potential of the luminol. However, ator above the redox potential of luminol, the luminol then transfers anelectron to the ETM, allowing rapid and repeated electron transfer. Inthis way, the electron source (or co-reductant) serves to amplify thesignal generated in the system, as the electron source molecules rapidlyand repeatedly donate electrons to the ETM of the label probe.

Luminol has the added benefit of becoming a chemiluminiscent speciesupon oxidation (see Jirka et al., Analytica Chimica Acta 284:345(1993)), thus allowing photo-detection of electron transfer from the ETMto the electrode. Thus, as long as the luminol is unable to contact theelectrode directly, i.e. in the presence of the SAM such that there isno efficient electron transfer pathway to the electrode, luminol canonly be oxidized by transferring an electron to the ETM on the labelprobe. When the ETM is not present, i.e. when the target sequence is nothybridized to the composition of the invention, luminol is notsignificantly oxidized, resulting in a low photon emission and thus alow (if any) signal from the luminol. In the presence of the target, amuch larger signal is generated. Thus, the measure of luminol oxidationby photon emission is an indirect measurement of the ability of the ETMto donate electrons to the electrode. Furthermore, since photondetection is generally more sensitive than electronic detection, thesensitivity of the system may be increased. Initial results suggest thatluminescence may depend on hydrogen peroxide concentration, pH, andluminol concentration, the latter of which appears to be non-linear.

Suitable electron source molecules are well known in the art, andinclude, but are not limited to, ferricyanide, and luminol.

Alternatively, output electron acceptors or sinks could be used, i.e.the above reactions could be run in reverse, with the ETM such as ametallocene receiving an electron from the electrode, converting it tothe metallicenium, with the output electron acceptor then accepting theelectron rapidly and repeatedly. In this embodiment, cobalticenium isthe preferred ETM.

The presence of the ETMs at the surface of the monolayer can be detectedin a variety of ways. A variety of detection methods may be used,including, but not limited to, optical detection (as a result ofspectral changes upon changes in redox states), which includesfluorescence, phosphorescence, luminiscence, chemiluminescence,electrochemiluminescence, and refractive index; and electronicdetection, including, but not limited to, amperommetry, voltammetry,capacitance and impedence. These methods include time or frequencydependent methods based on AC or DC currents, pulsed methods, lock-intechniques, filtering (high pass, low pass, band pass), andtime-resolved techniques including time-resolved fluoroscence.

In one embodiment, the efficient transfer of electrons from the ETM tothe electrode results in stereotyped changes in the redox state of theETM. With many ETMs including the complexes of ruthenium containingbipyridine, pyridine and imidazole rings, these changes in redox stateare associated with changes in spectral properties. Significantdifferences in absorbance are observed between reduced and oxidizedstates for these molecules. See for example Fabbrizzi et al., Chem. Soc.Rev. 1995 ppl 97-202). These differences can be monitored using aspectrophotometer or simple photomultiplier tube device.

In this embodiment, possible electron donors and acceptors include allthe derivatives listed above for photoactivation or initiation.Preferred electron donors and acceptors have characteristically largespectral changes upon oxidation and reduction resulting in highlysensitive monitoring of electron transfer. Such examples includeRu(NH₃)₄py and Ru(bpy)₂im as preferred examples. It should be understoodthat only the donor or acceptor that is being monitored by absorbanceneed have ideal spectral characteristics.

In a preferred embodiment, the electron transfer is detectedfluorometrically. Numerous transition metal complexes, including thoseof ruthenium, have distinct fluorescence properties. Therefore, thechange in redox state of the electron donors and electron acceptorsattached to the nucleic acid can be monitored very sensitively usingfluorescence, for example with Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺. Theproduction of this compound can be easily measured using standardfluorescence assay techniques. For example, laser induced fluorescencecan be recorded in a standard single cell fluorimeter, a flow through“on-line” fluorimeter (such as those attached to a chromatographysystem) or a multi-sample “plate-reader” similar to those marketed for96-well immuno assays.

Alternatively, fluorescence can be measured using fiber optic sensorswith nucleic acid probes in solution or attached to the fiber optic.Fluorescence is monitored using a photomultiplier tube or other lightdetection instrument attached to the fiber optic. The advantage of thissystem is the extremely small volumes of sample that can be assayed.

In addition, scanning fluorescence detectors such as the Fluorlmagersold by Molecular Dynamics are ideally suited to monitoring thefluorescence of modified nucleic acid molecules arrayed on solidsurfaces. The advantage of this system is the large number of electrontransfer probes that can be scanned at once using chips covered withthousands of distinct nucleic acid probes.

Many transition metal complexes display fluorescence with large Stokesshifts. Suitable examples include bis- and trisphenanthroline complexesand bis- and trisbipyridyl complexes of transition metals such asruthenium (see Juris, A., Balzani, V., et. al. Coord. Chem. Rev., V. 84,p. 85-277, 1988). Preferred examples display efficient fluorescence(reasonably high quantum yields) as well as low reorganization energies.These include Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺,Ru(4,4′-diphenyl-2,2′-bipyridine)₃ ²⁺ and platinum complexes (seeCummings et al., J. Am. Chem. Soc. 118:1949-1960 (1996), incorporated byreference). Alternatively, a reduction in fluorescence associated withhybridization can be measured using these systems.

In a further embodiment, electrochemiluminescence is used as the basisof the electron transfer detection. With some ETMs such as Ru²⁺ (bpy)₃,direct luminescence accompanies excited state decay. Changes in thisproperty are associated with nucleic acid hybridization and can bemonitored with a simple photomultiplier tube arrangement (see Blackburn,G. F. Clin. Chem. 37: 1534-1539 (1991); and Juris et al., supra.

In a preferred embodiment, electronic detection is used, includingamperommetry, voltammetry, capacitance, and impedence. Suitabletechniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltametry (cyclic voltametry, pulse voltametry(normal pulse voltametry, square wave voltametry, differential pulsevoltametry, Osteryoung square wave voltametry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time-dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement; capacitancemeasurement; AC voltametry; and photoelectrochemistry.

In a preferred embodiment, monitoring electron transfer is viaamperometric detection. This method of detection involves applying apotential (as compared to a separate reference electrode) between thenucleic acid-conjugated electrode and a reference (counter) electrode inthe sample containing target genes of interest. Electron transfer ofdiffering efficiencies is induced in samples in the presence or absenceof target nucleic acid; that is, the presence or absence of the targetnucleic acid, and thus the label probe, can result in differentcurrents.

The device for measuring electron transfer amperometrically involvessensitive current detection and includes a means of controlling thevoltage potential, usually a potentiostat. This voltage is optimizedwith reference to the potential of the electron donating complex on thelabel probe. Possible electron donating complexes include thosepreviously mentioned with complexes of iron, osmium, platinum, cobalt,rhenium and ruthenium being preferred and complexes of iron being mostpreferred.

In a preferred embodiment, alternative electron detection modes areutilized. For example, potentiometric (or voltammetric) measurementsinvolve non-faradaic (no net current flow) processes and are utilizedtraditionally in pH and other ion detectors. Similar sensors are used tomonitor electron transfer between the ETM and the electrode. Inaddition, other properties of insulators (such as resistance) and ofconductors (such as conductivity, impedance and capicitance) could beused to monitor electron transfer between ETM and the electrode.Finally, any system that generates a current (such as electron transfer)also generates a small magnetic field, which may be monitored in someembodiments.

It should be understood that one benefit of the fast rates of electrontransfer observed in the compositions of the invention is that timeresolution can greatly enhance the signal-to-noise results of monitorsbased on absorbance, fluorescence and electronic current. The fast ratesof electron transfer of the present invention result both in highsignals and stereotyped delays between electron transfer initiation andcompletion. By amplifying signals of particular delays, such as throughthe use of pulsed initiation of electron transfer and “lock-in”amplifiers of detection, and Fourier transforms.

In a preferred embodiment, electron transfer is initiated usingalternating current (AC) methods. Without being bound by theory, itappears that ETMs, bound to an electrode, generally respond similarly toan AC voltage across a circuit containing resistors and capacitors.Basically, any methods which enable the determinaton of the nature ofthese complexes, which act as a resistor and capacitor, can be used asthe basis of detection. Surprisingly, traditional electrochemicaltheory, such as exemplified in Laviron et al., J. Electroanal. Chem.97:135 (1979) and Laviron et al., J. Electroanal. Chem. 105:35 (1979),both of which are incorporated by reference, do not accurately model thesystems described herein, except for very small E_(AC) (less than 10 mV)and relatively large numbers of molecules. That is, the AC current (I)is not accurately described by Laviron's equation. This may be due inpart to the fact that this theory assumes an unlimited source and sinkof electrons, which is not true in the present systems.

The AC voltametry theory that models these systems well is outlined inO'Connor et al., J. Electroanal. Chem. 466(2):197-202 (1999), herebyexpressly incorporated by reference. The equation that predicts thesesystems is shown below as Equation 1: $\begin{matrix}\begin{matrix}{i_{avg} = {2{{nfFN}_{total} \cdot}}} \\{\frac{\sinh\left\lbrack {\frac{n\quad F}{RT} \cdot E_{AC}} \right\rbrack}{{\cosh\left\lbrack {\frac{nF}{RT} \cdot E_{AC}} \right\rbrack} + {\cosh\left\lbrack {\frac{nF}{RT}\left( {E_{DC} - E_{O}} \right)} \right\rbrack}}}\end{matrix} & {{Equation}\quad 1}\end{matrix}$

In Equation 1, n is the number of electrons oxidized or reduced perredox molecule, f is the applied frequency, F is Faraday's constant,N_(total) is the total number of redox molecules, E_(O) is the formalpotential of the redox molecule, R is the gas constant, T is thetemperature in degrees Kelvin, and E_(DC) is the electrode potential.The model fits the experimental data very well. In some cases thecurrent is smaller than predicted, however this has been shown to becaused by ferrocene degradation which may be remedied in a number ofways.

In addition, the faradaic current can also be expressed as a function oftime, as shown in Equation 2:

Equation 2

I_(F) is the Faradaic current and q_(e) is the elementary charge.

However, Equation 1 does not incorporate the effect of electron transferrate nor of instrument factors. Electron transfer rate is important whenthe rate is close to or lower than the applied frequency. Thus, the truei_(AC) should be a function of all three, as depicted in Equation 3.

Equation 3

-   -   i_(AC)=f(Nemst factors)f(k_(ET))f(instrument factors)

These equations can be used to model and predict the expected ACcurrents in systems which use input signals comprising both AC and DCcomponents. As outlined above, traditional theory surprisingly does notmodel these systems at all, except for very low voltages.

In general, non-specifically bound label probes/ETMs show differences inimpedance (i.e. higher impedances) than when the label probes containingthe ETMs are specifically bound in the correct orientation. In apreferred embodiment, the non-specifically bound material is washedaway, resulting in an effective impedance of infinity. Thus, ACdetection gives several advantages as is generally discussed below,including an increase in sensitivity, and the ability to “filter out”background noise. In particular, changes in impedance (including, forexample, bulk impedance) as between non-specific binding ofETM-containing probes and target-specific assay complex formation may bemonitored.

Accordingly, when using AC initiation and detection methods, thefrequency response of the system changes as a result of the presence ofthe ETM. By “frequency response” herein is meant a modification ofsignals as a result of electron transfer between the electrode and theETM. This modification is different depending on signal frequency. Afrequency response includes AC currents at one or more frequencies,phase shifts, DC offset voltages, faradaic impedance, etc.

Once the assay complex including the target sequence and label probe ismade, a first input electrical signal is then applied to the system,preferably via at least the sample electrode (containing the complexesof the invention) and the counter electrode, to initiate electrontransfer between the electrode and the ETM. Three electrode systems mayalso be used, with the voltage applied to the reference and workingelectrodes. The first input signal comprises at least an AC component.The AC component may be of variable amplitude and frequency. Generally,for use in the present methods, the AC amplitude ranges from about 1 mVto about 1.1 V, with from about 10 mV to about 800 mV being preferred,and from about 10 mV to about 500 mV being especially preferred. The ACfrequency ranges from about 0.01 Hz to about 100 MHz, with from about 10Hz to about 10 MHz being preferred, and from about 100 Hz to about 20MHz being especially preferred.

The use of combinations of AC and DC signals gives a variety ofadvantages, including surprising sensitivity and signal maximization.

In a preferred embodiment, the first input signal comprises a DCcomponent and an AC component. That is, a DC offset voltage between thesample and counter electrodes is swept through the electrochemicalpotential of the ETM (for example, when ferrocene is used, the sweep isgenerally from 0 to 500 mV) (or alternatively, the working electrode isgrounded and the reference electrode is swept from 0 to −500 mV). Thesweep is used to identify the DC voltage at which the maximum responseof the system is seen. This is generally at or about the electrochemicalpotential of the ETM. Once this voltage is determined, either a sweep orone or more uniform DC offset voltages may be used. DC offset voltagesof from about −1 V to about +1.1 V are preferred, with from about −500mV to about +800 mV being especially preferred, and from about −300 mVto about 500 mV being particularly preferred. In a preferred embodiment,the DC offset voltage is not zero. On top of the DC offset voltage, anAC signal component of variable amplitude and frequency is applied. Ifthe ETM is present, and can respond to the AC perturbation, an ACcurrent will be produced due to electron transfer between the electrodeand the ETM.

For defined systems, it may be sufficient to apply a single input signalto differentiate between the presence and absence of the ETM (i.e. thepresence of the target sequence) nucleic acid. Alternatively, aplurality of input signals are applied. As outlined herein, this maytake a variety of forms, including using multiple frequencies, multipleDC offset voltages, or multiple AC amplitudes, or combinations of any orall of these.

Thus, in a preferred embodiment, multiple DC offset voltages are used,although as outlined above, DC voltage sweeps are preferred. This may bedone at a single frequency, or at two or more frequencies

In a preferred embodiment, the AC amplitude is varied. Without beingbound by theory, it appears that increasing the amplitude increases thedriving force. Thus, higher amplitudes, which result in higheroverpotentials give faster rates of electron transfer. Thus, generally,the same system gives an improved response (i.e. higher output signals)at any single frequency through the use of higher overpotentials at thatfrequency. Thus, the amplitude may be increased at high frequencies toincrease the rate of electron transfer through the system, resulting ingreater sensitivity. In addition, this may be used, for example, toinduce responses in slower systems such as those that do not possessoptimal spacing configurations.

In a preferred embodiment, measurements of the system are taken at atleast two separate amplitudes or overpotentials, with measurements at aplurality of amplitudes being preferred. As noted above, changes inresponse as a result of changes in amplitude may form the basis ofidentification, calibration and quantification of the system. Inaddition, one or more AC frequencies can be used as well.

In a preferred embodiment, the AC frequency is varied. At differentfrequencies, different molecules respond in different ways. As will beappreciated by those in the art, increasing the frequency generallyincreases the output current. However, when the frequency is greaterthan the rate at which electrons may travel between the electrode andthe ETM, higher frequencies result in a loss or decrease of outputsignal. At some point, the frequency will be greater than the rate ofelectron transfer between the ETM and the electrode, and then the outputsignal will also drop.

In one embodiment, detection utilizes a single measurement of outputsignal at a single frequency. That is, the frequency response of thesystem in the absence of target sequence, and thus the absence of labelprobe containing ETMs; can be previously determined to be very low at aparticular high frequency. Using this information, any response at aparticular frequency, will show the presence of the assay complex. Thatis, any response at a particular frequency is characteristic of theassay complex. Thus, it may only be necessary to use a single input highfrequency, and any changes in frequency response is an indication thatthe ETM is present, and thus that the target sequence is present.

In addition, the use of AC techniques allows the significant reductionof background signals at any single frequency due to entities other thanthe ETMs, i.e. “locking out” or “filtering” unwanted signals. That is,the frequency response of a charge carrier or redox active molecule insolution will be limited by its diffusion coefficient and chargetransfer coefficient. Accordingly, at high frequencies, a charge carriermay not diffuse rapidly enough to transfer its charge to the electrode,and/or the charge transfer kinetics may not be fast enough. This isparticularly significant in embodiments that do not have goodmonolayers, i.e. have partial or insufficient monolayers, i.e. where thesolvent is accessible to the electrode. As outlined above, in DCtechniques, the presence of “holes” where the electrode is accessible tothe solvent can result in solvent charge carriers “short circuiting” thesystem, i.e. the reach the electrode and generate background signal.However, using the present AC techniques, one or more frequencies can bechosen that prevent a frequency response of one or more charge carriersin solution, whether or not a monolayer is present. This is particularlysignificant since many biological fluids such as blood containsignificant amounts of redox active molecules which can interfere withamperometric detection methods.

In a preferred embodiment, measurements of the system are taken at atleast two separate frequencies, with measurements at a plurality offrequencies being preferred. A plurality of frequencies includes a scan.For example, measuring the output signal, e.g., the AC current, at a lowinput frequency such as 1-20 Hz, and comparing the response to theoutput signal at high frequency such as 10-100 kHz will show a frequencyresponse difference between the presence and absence of the ETM. In apreferred embodiment, the frequency response is determined at at leasttwo, preferably at least about five, and more preferably at least aboutten frequencies.

After transmitting the input signal to initiate electron transfer, anoutput signal is received or detected. The presence and magnitude of theoutput signal will depend on a number of factors, including theoverpotential/amplitude of the input signal; the frequency of the inputAC signal; the composition of the intervening medium; the DC offset; theenvironment of the system; the nature of the ETM; the solvent; and thetype and concentration of salt. At a given input signal, the presenceand magnitude of the output signal will depend in general on thepresence or absence of the ETM, the placement and distance of the ETMfrom the surface of the monolayer and the character of the input signal.In some embodiments, it may be possible to distinguish betweennon-specific binding of label probes and the formation of targetspecific assay complexes containing label probes, on the basis ofimpedance.

In a preferred embodiment, the output signal comprises an AC current. Asoutlined above, the magnitude of the output current will depend on anumber of parameters. By varying these parameters, the system may beoptimized in a number of ways.

In general, AC currents generated in the present invention range fromabout 1 femptoamp to about 1 milliamp, with currents from about 50femptoamps to about 100 microamps being preferred, and from about 1picoamp to about 1 microamp being especially preferred.

In a preferred embodiment, the output signal is phase shifted in the ACcomponent relative to the input signal. Without being bound by theory,it appears that the systems of the present invention may be sufficientlyuniform to allow phase-shifting based detection. That is, the complexbiomolecules of the invention through which electron transfer occursreact to the AC input in a homogeneous manner, similar to standardelectronic components, such that a phase shift can be determined. Thismay serve as the basis of detection between the presence and absence ofthe ETM, and/or differences between the presence of target-specificassay complexes comprising label probes and non-specific binding of thelabel probes to the system components.

The output signal is characteristic of the presence of the ETM; that is,the output signal is characteristic of the presence of thetarget-specific assay complex comprising label probes and ETMs. In apreferred embodiment, the basis of the detection is a difference in thefaradaic impedance of the system as a result of the formation of theassay complex. Faradaic impedance is the impedance of the system betweenthe electrode and the ETM. Faradaic impedance is quite different fromthe bulk or dielectric impedance, which is the impedance of the bulksolution between the electrodes. Many factors may change the faradaicimpedance which may not effect the bulk impedance, and vice versa. Thus,the assay complexes comprising the nucleic acids in this system have acertain faradaic impedance, that will depend on the distance between theETM and the electrode, their electronic properties, and the compositionof the intervening medium, among other things. Of importance in themethods of the invention is that the faradaic impedance between the ETMand the electrode is signficantly different depending on whether thelabel probes containing the ETMs are specifically or non-specificallybound to the electrode.

Accordingly, the present invention further provides apparatus for thedetection of nucleic acids using AC detection methods. The apparatusincludes a test chamber which has at least a first measuring or sampleelectrode, and a second measuring or counter electrode. Three electrodesystems are also useful. The first and second measuring electrodes arein contact with a test sample receiving region, such that in thepresence of a liquid test sample, the two electrodes may be inelectrical contact.

In a preferred embodiment, the first measuring electrode comprises asingle stranded nucleic acid capture probe covalently attached via anattachment linker, and a monolayer comprising conductive oligomers, suchas are described herein.

The apparatus further comprises an AC voltage source electricallyconnected to the test chamber; that is, to the measuring electrodes.Preferably, the AC voltage source is capable of delivering DC offsetvoltage as well.

In a preferred embodiment, the apparatus further comprises a processorcapable of comparing the input signal and the output signal. Theprocessor is coupled to the electrodes and configured to receive anoutput signal, and thus detect the presence of the target nucleic acid.

Thus, the compositions of the present invention may be used in a varietyof research, clinical, quality control, or field testing settings.

In a preferred embodiment, the probes are used in genetic diagnosis. Forexample, probes can be made using the techniques disclosed herein todetect target sequences such as the gene for nonpolyposis colon cancer,the BRCA1 breast cancer gene, P53, which is a gene associated with avariety of cancers, the Apo E4 gene that indicates a greater risk ofAlzheimer's disease, allowing for easy presymptomatic screening ofpatients, mutations in the cystic fibrosis gene, or any of the otherswell known in the art.

In an additional embodiment, viral and bacterial detection is done usingthe complexes of the invention. In this embodiment, probes are designedto detect target sequences from a variety of bacteria and viruses. Forexample, current blood-screening techniques rely on the detection ofanti-HIV antibodies. The methods disclosed herein allow for directscreening of clinical samples to detect HIV nucleic acid sequences,particularly highly conserved HIV sequences. In addition, this allowsdirect monitoring of circulating virus within a patient as an improvedmethod of assessing the efficacy of anti-viral therapies. Similarly,viruses associated with leukemia, HTLV-I and HTLV-II, may be detected inthis way. Bacterial infections such as tuberculosis, clymidia and othersexually transmitted diseases, may also be detected, for example usingribosomal RNA (rRNA) as the target sequences.

In a preferred embodiment, the nucleic acids of the invention find useas probes for toxic bacteria in the screening of water and food samples.For example, samples may be treated to lyse the bacteria to release itsnucleic acid (particularly rRNA), and then probes designed to recognizebacterial strains, including, but not limited to, such pathogenicstrains as, Salmonella, Campylobacter, Vibrio cholerae, Leishmania,enterotoxic strains of E. coli and Legionnaire's disease bacteria.Similarly, bioremediation strategies may be evaluated using thecompositions of the invention.

In a further embodiment, the probes are used for forensic “DNAfingerprinting” to match crime-scene DNA against samples taken fromvictims and suspects.

In an additional embodiment, the probes in an array are used forsequencing by hybridization.

Thus, the present invention provides for extremely specific andsensitive probes, which may, in some embodiments, detect targetsequences without removal of unhybridized probe. This will be useful inthe generation of automated gene probe assays.

Alternatively, the compositions of the invention are useful to detectsuccessful gene amplification in PCR, thus allowing successful PCRreactions to be an indication of the presence or absence of a targetsequence. PCR may be used in this manner in several ways. For example,in one embodiment, the PCR reaction is done as is known in the art, andthen added to a composition of the invention comprising the targetnucleic acid with a ETM, covalently attached to an electrode via aconductive oligomer with subsequent detection of the target sequence.Alternatively, PCR is done using nucleotides labelled with a ETM, eitherin the presence of, or with subsequent addition to, an electrode with aconductive oligomer and a target nucleic acid. Binding of the PCRproduct containing ETMs to the electrode composition will allowdetection via electron transfer. Finally, the nucleic acid attached tothe electrode via a conductive polymer may be one PCR primer, withaddition of a second primer labelled with an ETM. Elongation results indouble stranded nucleic acid with a ETM

and electrode covalently attached. In this way, the present invention isused for PCR detection of target sequences.

In a preferred embodiment, the arrays are used for mRNA detection. Apreferred embodiment utilizes either capture probes or capture extenderprobes that hybridize close to the 3′ polyadenylation tail of the mRNAs.This allows the use of one species of target binding probe fordetection, i.e. the probe contains a poly-T portion that will bind tothe poly-A tail of the mRNA target. Generally, the probe will contain asecond portion, preferably non-poly-T, that will bind to the detectionprobe (or other probe). This allows one target-binding probe to be made,and thus decreases the amount of different probe synthesis that is done.

In a preferred embodiment, the use of restriction enzymes and ligationmethods allows the creation of “universal” arrays. In this embodiment,monolayers comprising capture probes that comprise restrictionendonuclease ends, as is generally depicted in FIG. 7 of PCT US97/20014. By utilizing complementary portions of nucleic acid, while leaving“sticky ends”, an array comprising any number of restrictionendonuclease sites is made. Treating a target sample with one or more ofthese restriction endonucleases allows the targets to bind to the array.This can be done without knowing the sequence of the target. The targetsequences can be ligated, as desired, using standard methods such asligases, and the target sequence detected, using either standard labelsor the methods of the invention.

The present invention provides methods which can result in sensitivedetection of nucleic acids. In a preferred embodiment, less than about10×10⁶ molecules are detected, with less than about 10×10⁵ beingpreferred, less than 10×10⁴ being particularly preferred, less thanabout 10×10³ being especially preferred, and less than about 10×10²being most preferred. As will be appreciated by those in the art, thisassumes a 1:1 correlation between target sequences and reportermolecules; if more than one reporter molecule (i.e. electron transfermoeity) is used for each target sequence, the sensitivity will go up.

While the limits of detection are currently being evaluated, based onthe published electron transfer rate through DNA, which is roughly10×10⁶ electrons/sec/duplex for an 8 base pair separation (see Meade etal., Angw. Chem. Eng. Ed., 34:352 (1995)) and high driving forces, ACfrequencies of about 100 kHz should be possible. As the preliminaryresults show, electron transfer through these systems is quiteefficient, resulting in nearly 100×10³ electrons/sec, resulting inpotential femptoamp sensitivity for very few molecules.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference.

EXAMPLES Example 1 Preparation and Evaluation of Asymmetric Monolayers

PreDaration of Asymmetric Monolayers

Synthesis of Symmetrical Disulfide

Symmetrical disulfides may be prepared from neopentyl alcohol or fromneopentyl iodide in the presence of thiol acetic acid. Preferably, thereaction is carried out using neopentyl alcohol in the presence of PPh3and DIPAD. The resulting disulfide is recrystallized from hexane,purified on a chromatographic column, and treated with sodium hydroxideto yield a symmetrical disulfide (See FIG. 3A).

Synthesis of CT103

The strategy for the synthesis of a precursor for CPG with an AMFS wasbased on the chemistry established for exchanging an symmetricaldisulfide with acetyl thiol moiety in the presence of a base as shown inFIG. 3C. First, CT100 was obtained from dimethyloxetane and thiolaceticacide. Next, the symmetrical disulfide CT101 was obtained from CT100 bytreating CT100 with NaOH and dioxane. Treatment of K133 and CT101 in thepresence of NaOH and Dioxane yielded the asymmetrical monolayer formingspecies CT102. Further treatment of CT102 with succinic anhydride

Synthesis of CT105

The synthesis of CT105 is depicted in FIG. 3E and involves the followingsteps:

Synthesis of M41. To a flask was added sodium hydride (6.68 g, 60% inmineral oil) to a solution of triethylene glycol (83.7 g) in 1 Lanhydrous DMF. The reaction mixture was stirred for 45 min at roomtemperature, then cool to 0° C. in an ice bath. To the mixture was added1 1-bromo-1-undecene (26 g) in 100 mL of DMF dropwise within 30 min at0° C. and then the mixture was vigorously stirred for 1 hour at 0° C.After removing the cooling bath, the reaction mixture was vigorouslystirred overnight at room temperature. To the mixture was add 50 mL ofwater, then the solution was concentrated on a high vacuum rotavapor. Tothe residue was add 400 mL of ethyl acetate and 600 mL of water and theorganic layer was separated, and the aqueous layer was extracted with30% ethyl acetate/70% hexane solution (3×300 mL). The combined organiclayers were washed with water (2×300 ml), dried over anhydrous sodiumsulfate, filtered and concentrated. T he residue was purified withsilica gel chromatography eluted with 10%-70% ethyl acetate in hexane toprovide the desired product.

Synthesis of M42. To a Shlenk tube were added M41 (7.6 g), thioaceticacid (2.2 mL), and t-butyl peroxide (0.48 mL). Then the solution wasflushed with argon for 5 min. After the cap was closed, the tube washeated at 100° C. overnight in an oil bath. The tube was cooled to roomtemperature, and the reaction mixture was diluted with 400 mL ofdichloromethane. The organic solution was washed with 200 mL of 5%sodium bicarbonate. Then the aqueous layer was extracted withdichloromethane (2×300 mL). The combined organic layers, was washed withbrine, dried over anhydrous sodium sulfate, filtered and concentrated.The residue was purified with silica gel chromatography eluted with30%-50% ethyl acetate in hexane to provide the desired product.

Synthesis of CT101. To a flask was added 3,3-dimethyloxetane (8.25 g, 96mmol) and thiol acetic acid (13.06 g, 172 mmol). The reaction mixturewas heated to 65° C. in an oil bath for 40 hours. Then the non-consumedstarting material was removed by distillation under vacuum (20-30 mmHg)at 65° C. The crude product was dissolved in 60 mL of methanol in around bottom flask and potassium carbonate (15.6 g) was added. Thereaction mixture was vigorously stirred at room temperature in open airfor 24 hours. The reaction mixture was filtered through a bed of Celiteand washed with a mixture of methanol and dichloromethane (2:1, 3×50mL). Then the filtrate was concentrated. The residue was purified withsilica gel chromatography eluted with 1% -5% methanol in dichloromethaneto provide the desired product. ¹H NMR (300 MHz, CDCl₃)_(—)3.48 (s, 2H,CH₂O), 2.89 (s, 2H, CH₂S), 1.01 (s, 6H, 2×CH ₃).

Synthesis of CT105. To a flask containing M42 (0.64 g) and CT101(1.0 g)in THF (10 mL) and methanol (10 mL) was added NaOH solution (1.0 mL, 8M). The mixture was stirred in the air for six hours. Then the solventswere removed on a rotavapor at 40° C. and residue was dissolved in 150mL of dichloromethane. The mixture was extracted with water (2×50 mL)and the organic layer dried over anhydrous sodium sulfate, filtered andconcentrated. The residue was purified with silica gel chromatographyeluted with 50% -90% ethyl acetate in hexane to provide the desiredproduct as colorless viscous oil. ¹H NMR (300 MHz, CDCl₃)_(—)3.80-3.62(m, 12H, 6×OCH₂), 3.47 (m, 4H, 2×CH₂OH), 2.85 (s, 2H, SCH₂CMe₂), 2.74(t, J=7.2 Hz, 2H, SCH₂), 1.77-1.63 (m, 4H, 2×CH₂), 1.32 (m, 16H), 1.02(s, 6H, CMe₂). Anal. calcd. for (C₂₂H₄₆O₅S₂ +Na)⁺: 477. Found: 477.

Electrochemical Evaluation of New Insulators

The following oligonucleotides were used to evaluate the new insulators:

1) Capture oligonucleotides: D1650: 5′-TCA TTG ATG GTC TCT TTT AACA(N152) D1678: 5′-GAC TGA CTC GTA CTA(N152)

2) Direct assay signaling probe: D1085: 5′-TCT ACA G(N6) C(N6) TGT TAAAAG AGA CCA TCA ATG A

3) Sandwich Assay target and signaling probes: D765: 5′-GAC ATC AAG CAGCCA TGC AAA TGT TAA AAG AGA CCA TCA ATG AGG AAG CTG CAG AAT GGG ATA GAGTGC ATC CAG T D772: 5′-(N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6)C(N6) G(C131) TTT GCA TGG CTG CTT GAT GTC D1156: 5′-CAC AGT GGG GGG ACATCA AGC AGC CAT GCA AAT GTT AAA AGA GAC CAT CAA TGA GGA AGC TGC AGA ATGGGA TAG AGT CAT CCA GT

D1355 (20 Fc), D1356 (30 Fc) and D1357 (36 Fc) have the similar sequenceas D1358 (54 (Fc): D1358: 5′-(C23)(C23)(C23)(C23)(C23)(C23)(N87)(N87)(N87)(N87) ATC (C140)(N87)(N 87)(N87)(N87)(C140) TTT GCA TGG CTG CTT GATGTC CCC CCA CTG TG D998: 5′-TGT GCA GTT GAC GTG GAT TGT TAA AAG AGA CCATCA ATG AGG AAG CTG GAG AAT GGG ATA GAG TCA TCC AGT D1055 (20Fc):5′-(C23)(C23)(C23)(C23)(N87)(N87) (N87)(N87)(C140) ATC CAC GTG AAC TGGAGAChip preparation and deposition solution.

Chips were made on spotting machine. To diminish the effect of chipmaterials, the chips with different insulators were made from the samecircuit board. An array chip (lot # DC228, DC229, and DC231, TypeCB37-4) with sensor pads containing self assembled monolayer accordingto

the pattern shown in FIG. 10.

The pad surfaces were respectively treated with the depositionsolutions. To reduce the influence of the concentration of captureoligonucleotides, a stock deposition solution was prepared accordingtothe standard procedure without insulator. Next, the stock solution wasdivided into three portions, into which the insulators, M44, CT99 andCT105, were added respectively. The final deposition solutions consistedof a mixture of DNA/H6-two-unit wire/insulator (with ratio of 1/10/5)with a total thiol concentration of 53 μM. The pads are deposited usingthe spotting machine and post treated with insulator. As shown in FIG.10, the chip has two rows of capture pads (D1650), a row of negativecontrol pads (D1678) non-specific binding pads (D1678), and a row ofinsulator. The DNA-probe of D1650 is complementary to the targetoligonucleotide D1085, D765, D998, and D1156. D1678 is non-complementaryto either target or signaling oligonucleotides.

Hybridization solution and testing.

For direct assays, the solution consists of 200 nM of signaling probeand 41% H₂O, 25% 4000 mM NaClO₄ with 80 mM Tris (pH 6.5) 1.0 mM C6insulator, 10% FCS, and 24% lysis buffer.

For sandwich assays, the hybridization solution consisted of 10 nMtarget oligonucleotides and 30 nM signaling molecule in the mixture of41% H ₂O, 25% 4000 nM NaClO₄ with 80 mM Tris, pH 6.5, 1.0 mM C6insulator, 10% FCS, and 24% lysis buffer.

The hybridization solution is injected into the cartridge of 3 chips.The solution is allowed to hybridize at room temperature for 2 hours fordirect assays or for overnight for sandwich assays. The chip is thenplugged into the reader and scanned in 4th harmonics at differentfrequencies.

All peaks are calculated using the auto peak finder. Ip of each data set(at least three chips) and the standard deviation at all of thefrequencies are calculated. The normalized frequency response isdetermined by Ip (high frequency)/Ip(10 Hz)×folds increase infrequency).

As shown in FIG. 11 (direct assay of 2 N6 ferrocene signaling probe),FIG. 12 (sandwich assay of 8 N6 ferrocene), FIG. 13 (sandwich assay of20 C23 type ferrocene) and FIG. 14 (sandwich assay for 54 C23 typeferrocene), the new insulators, CT99 and CT105, gave much betterelectrochemical response than control insulator M44. All data werecollected for 1000 Hz at 4^(th) harmonics.

However, the nonspecific binding of three different insulators issimilar. FIG. 15 and FIG. 16, illustrate nonspecific binding for directand sandwich assays at 1000 Hz and ₄th harmonics. Non-specific signalingon the pads with insulator alone was higher for the new insulators, CT99and CT105, but there were no difference on pads containing noncomplementary DNA.

FIG. 17 depicts a monolayer comprising insulators only (i.e. M44) and amonolayer comprising asymmetric monolayer forming species (i.e. CT105).

In order to further evaluate the behavior of the new insulators, afrequency response study was carried out at 10 Hz, 100 Hz and 100 Hz. Asshown in FIGS. 18-20, the new insulators gave better frequency responsethan the control insulator M44. FIG. 18 is the frequency response forD1085 of two N6 ferrocenes, while FIG. 19 and FIG. 20,respectively arethe frequency response for the sandwich assays of 8 and 20 ferrocenesystems.

1. A method of modifying a metallic surface comprising contacting themetallic surface with an asymmetric monolayer forming species having theformula:

wherein A is an attachment linker moiety; MFS is a monlayer formingspecies; and AG is an electroconduit forming species.
 2. A methodaccording to claim 1 further comprising contacting said metallic surfacewith a biological species having the formula: A-MFS-capture bindingligand wherein A is an attachment linker; and MFS is a monolayer formingspecies.
 3. A method according to claim 2 wherein said capture bindingligand is a nucleic acid.
 4. A method according to claim 2 wherein saidcapture binding ligand is a n protein.
 5. A method according to claim 1wherein A is sulfur.
 6. A method according to claim 1 wherein saidmetallic surface is gold.
 7. A method according to claim 1 wherein saidMFS is an insulator.
 8. A method according to claim 7 wherein saidinsulator comprises an alkyl group from about 7 to 20 carbons.
 9. Amethod according to claim 8 wherein said alkyl group comprises aheteroalkyl.
 10. A method according to claim 8 wherein said alkyl groupcomprises a substituted alkyl.
 11. A method according to claim 1 whereinsaid AG comprises an alkyl group from about 1 to 6 carbons.
 12. A methodaccording to claim 1 or 11 wherein said AG is branched, having theformula:

wherein R₃ through R₅ are independently selected from the groupconsisting of hydrogen, alkyl, aryl, alcohol, amine, amido, nitro,ether, ester, ketone, imino, aldehyde, alkoxy, carbonyl, halogen, sulfurcontaining moiety and phosphorus containing moiety;
 13. A methodaccording to claim 12 wherein said AG is attached to said attachmentlinker via a (CH₂)_(n) group, wherein n is an integer from 0 to
 4. 14. Amethod according to claim 12 wherein said AG is attached directly tosaid attachment linker.